Chardonnay grape seed products for nonalcoholic fatty liver disease

ABSTRACT

Embodiments described herein provide for uses of grape products, such as Chardonnay grape products and grape seed flour, for use in promoting a healthy liver, promoting healthy fat content in a liver, for treating or preventing NAFLD, and other embodiments and uses as described herein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a National Stage filing under 35 U.S.C 371 of PCTApplication No. PCT/US15/66585, filed Dec. 18, 2015, which claims thebenefit of priority to U.S. Provisional Application No. 62/094,963,filed Dec. 20, 2014, each of which are incorporated by reference hereinin their entirety.

FIELD

Embodiments described herein provide for uses of grape products,including, but not limited to Chardonnay grape products.

BACKGROUND

Nonalcoholic fatty liver disease (NAFLD) is recognized as a significantpublic health problem. Prevalence of NAFLD is 20% to 30% of the generalpopulation of Western countries. NAFLD ranges from steatosis (simplefatty liver) to nonalcoholic steatohepatitis (NASH), a condition thatincreases liver-related morbidity and mortality. Excessive hepatic lipidaccumulation, oxidative and endoplasmic reticulum (ER) stress, andinsulin resistance are the major manifestations of the progression ofthis disease. Without being bound to any particular theory, NAFLD can becaused, for example, by excessive hepatic lipid accumulation followed byincreased oxidative stress and inflammation results in liver damage.NAFLD is a continuing problem. Embodiments described herein provide forsolutions to these problems and others.

SUMMARY

Embodiments disclosed herein provide methods of treating or preventingnon-alcoholic fatty liver disease (NAFLD) in a mammal comprisingadministering to the mammal an amount of a grape seed product effectiveto treat or prevent NAFLD.

Embodiments disclosed herein provide methods of reducing hepaticsteatosis in a mammal comprising administering to the mammal an amountof a grape product effective to reduce hepatic steatosis.

Embodiments disclosed herein provide methods of reducing steatohepatitisin a mammal comprising administering to the mammal an amount of a grapeproduct effective to reduce steatohepatitis.

Embodiments disclosed herein provide methods of reducing hepaticfibrosis in a mammal comprising administering to the mammal an amount ofa grape product effective to reduce hepatic fibrosis.

Embodiments disclosed herein provide methods of treating or preventingnon-alcoholic steatohepatitis (NASH) in a mammal comprisingadministering to the mammal an amount of a grape product effective totreat or prevent NASH.

Embodiments disclosed herein provide methods of supporting a healthyliver in a mammal comprising administering to the mammal an amount of agrape product effective to support a healthy liver.

In some embodiments of the methods described herein, the mammal has beenidentified as having non-alcoholic fatty liver disease. In someembodiments, the grape product reduces the amount of fat deposited inthe liver.

Embodiments described herein provide pharmaceutical compositionscomprising a grape product and at least one additional compound.

Embodiments disclosed herein provide methods of modulating theexpression of one or more of the genes as described herein comprisingadministering a grape product to a mammal expressing one or more of thegenes.

In some embodiments, the grape product is a grape seed flour, wholepomace or a fraction thereof (e.g., fractions of seed, skin, leaf, stem,and the like), grape extract (e.g. seed extract, skin extract, and thelike), grape skin product (e.g., skin flower, skin powder, and thelike), or any combination thereof, or any extract thereof. In someembodiments, the grape product is a Chardonnay product, such as, but notlimited to a Chardonnay grape seed flour.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Effect of Chardonnay grape seed flour (ChrSd) on plasma lipids.Male diet-induced obese mice (DIO) were fed high-fat (HF) dietscontaining 5% microcrystalline cellulose (MCC, control) or 10% (w/w)ChrSd for 5 weeks, and blood was collected in a food-deprived state.VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; HDL,high-density lipoprotein. Data are expressed as means±SE; n=8-10/group.P<0.05.

FIGS. 2A and 2B. Panel A. Glucose tolerance in obese mice fed a high-fat(HF) diet supplemented with either 5% microcrystalline cellulose (MCC,control) or 10% (w/w) Chardonnay grape seed flour (ChrSd) for 5 weeks.(A) Glucose tolerance tests (GTT) were performed in the fasting state.Panel B Area under the curve (AUC) values (derived from A). Data areexpressed as mean±SE. n=8-9/group. P<0.05.

FIGS. 3A and 3B. Panel A. Insulin tolerance in obese mice fed a high-fat(HF) diet supplemented with either 5% microcrystalline cellulose (MCC,control) or 10% (w/w) Chardonnay grape seed flour (ChrSd) for 5 weeks.(A) Insulin tolerance tests (ITTs) were performed in the fasting state.Panel B. Area under the curve (AUC) values (derived from A). Data areexpressed as mean±SE. n=8-9/group. P<0.05.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing, suitablemethods and materials are described herein. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. Other features and advantages of theembodiments disclosed herein will be apparent from the present detaileddescription and claims.

The term “salt” or “salts” may refer to any acid addition salts,including addition salts of free acids or addition salts of free bases.All of these salts (or other similar salts) may be prepared byconventional means. All such salts are acceptable provided that they arenon-toxic and do not substantially interfere with the desiredpharmacological activity.

The terms “pharmaceutically acceptable” and/or “therapeuticallyacceptable” refer to molecular entities and compositions that arephysiologically tolerable and preferably do not typically produce anallergic or similar untoward reaction, such as gastric upset, dizzinessand the like, when administered to a human. In some embodiments, as usedherein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a State government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia (e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985)) for use in animals, and more particularly in humans.

The term “about” or “approximately” means plus or minus 5%.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise.

As used herein the terms “comprise,” “have,” and “include” and theirconjugates, as used herein, mean “including but not limited to.” Whilevarious compositions, methods, and devices are described in terms of“comprising” various components or steps (interpreted as meaning“including, but not limited to”), the compositions, methods, and devicescan also “consist essentially of” or “consist of” the various componentsand steps.

As used herein, the phrase “in need thereof” means that the animal ormammal has been identified or suspected as having a need for theparticular method or treatment. In some embodiments, the identificationcan be by any means of diagnosis. In any of the methods and treatmentsdescribed herein, the animal or mammal can be in need thereof. In someembodiments, the animal or mammal is in an environment or will betraveling to an environment in which a particular disease, disorder, orcondition is prevalent.

As used herein, the term “subject,” “individual” or “patient,” usedinterchangeably, means any animal, including mammals, such as mice,rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses,or primates, such as humans.

As used herein, the term “animal” includes, but is not limited to,humans and non-human vertebrates such as wild, domestic, and farmanimals.

As used herein, the phrase “integer from X to Y” means any integer thatincludes the endpoints. That is, where a range is disclosed, eachinteger in the range including the endpoints is disclosed. For example,the phrase “integer from 1 to 5” discloses 1, 2, 3, 4, or 5 as well asthe range 1 to 5.

As used herein, the term “mammal” means a rodent (i.e., a mouse, a rat,or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or ahuman. In some embodiments, the mammal is a human.

As used herein, the terms “treat,” “treated,” or “treating” can refer totherapeutic treatment and/or prophylactic or preventative measureswherein the object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder or disease, or obtain beneficial ordesired clinical results. For purposes of the embodiments describedherein, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms; diminishment of extent ofcondition, disorder or disease; stabilized (i.e., not worsening) stateof condition, disorder or disease; delay in onset or slowing ofcondition, disorder or disease progression; amelioration of thecondition, disorder or disease state or remission (whether partial ortotal), whether detectable or undetectable; an amelioration of at leastone measurable physical parameter, not necessarily discernible by thepatient; or enhancement or improvement of condition, disorder ordisease. Treatment can also include eliciting a clinically significantresponse without excessive levels of side effects. Treatment alsoincludes prolonging survival as compared to expected survival if notreceiving treatment.

As described herein, grape products, such as Chardonnay grape products,may be used as a treatment for non-alcoholic fatty liver disease (NAFLD)and its more severe form, non-alcoholic steatohepatitis (NASH) inhumans. Other embodiments are described herein.

Embodiments described herein relate to uses of grape products. In someembodiments, the grape product is a Chardonnay grape product. Othergrape products are also described herein.

In some embodiments, grape product is a grape seed product. In someembodiments, the seed products contain the defatted portion of seed(e.g. grape seed), such as pomace meal, pomace flour, seed meal, or, insome embodiments, seed flour. In some embodiments, the seed productincludes the contents of defatted seeds that are not extractable by anorganic solvent, e.g., are not extractable by ethanol and/or methanol.In some embodiments, the seed product is prepared from seeds having anepicatechin content of at least 600 mg of epicatechin per 100 g of seedsor an epicatechin content of at least 700 mg of epicatechin per 100 g ofseeds. In some embodiments, the epicatechin content ranges from 600-800mg/100 g of seeds or from 650-800 mg/100 g of seeds.

As described herein, the grape product can be whole pomace or a portionthereof, grape skin product, grape seed product, or any combinationthereof, or any extract thereof. Each of the products or extractsthereof can be used alone or in any combination with one another. Theproducts or extracts can be derived from Chardonnay grapes, CabernetSauvignon grapes, Pinot Noir grape products, Sauvignon Blanc grapeproducts, White Riesling grape products, and the like, or anycombination thereof. In some embodiments, the grape is a grape grown ina coastal region. In some embodiments, the grape is a grape grown in aWinkler region climate type I, II, III, IV or V.

For the avoidance of doubt, a grape product is not a whole intact grape,but rather is a grape that has been processed. Examples of grapeproducts are described herein.

As used herein, “seed meal” is ground whole seeds and “seed flour” isground seed after the oil has been extracted. Seed flour may be obtainedusing the “cold press”, “hot press” and solvent extraction processes asare known in the art to extract the oil from seeds yielding defattedseed flour. The meal or flour can be dried to the desired moisturecontent using conventional drying techniques suitable for drying foodproducts.

The dried meal or flour can be further ground under ambient temperatureconditions to form seed powder having free-flowing particles. In someembodiments, the free-flowing particles can range from a size notexceeding 841 microns (20 mesh) to a size not exceeding 37 microns (400mesh). In certain embodiments, the size does not exceed 20 mesh, 40mesh, 60 mesh, 80 mesh, 100 mesh, 200 mesh, 300 mesh, or 400 mesh.

In some embodiments, seed flour is made by separating and drying grapeseeds, for example from the pomace produced after grapes are pressed toproduce grape juice (e.g., to make wine). The grape seeds can be“cold-pressed” to defat them (producing seed oil as a byproduct). Grapeseed flours are milled from the press cake after the oil is expelled. Inone embodiment, after juicing the grape the seed is separated from theskins, cleaned, mechanically defatted, finely milled and sifted tocreate an 100-80 mesh (149-177 micron) flowable powder.

Seed flour can also be purchased from a variety of sources. For example,certain types of grape seed flour can be purchased from Aprés Vin(Yakima, Wash.), Botanical Oil Innovations (Spooner, Wis.) orFruitsmart, Inc. (Grandview, Wash.). The seed flour can sometimes bepurchased as an 80 mesh flowable powder. In some embodiments, this isproduct is further milled and sifted to produce a flour with a smallerparticle size.

In some embodiments, skins, stems and leaves (the remainder of pomace)are removed from the seeds prior to pressing. Removal of the skins,stems, and leaves allows for optimal oil pressing.

In some embodiments, the grape product is whole grape pomace or aportion thereof including fractions of seed, skin, leaf, stem, and thelike. These fractions can also be present in a mixture of differentratios.

“Seed extract” can be made by solvent extraction of grape seeds with asuitable solvent, such as ethanol or methanol. For example, a grape seedextract, such as, but not limited to Chardonnay seed extract, can bemade using a 40% ethanol solution as the extraction solvent. Theextraction process, in addition to the extract containing the solventsoluble components, also produces a residue of non-soluble solids. Theseed extract can then be combined with other grape products describedherein or be used alone. Other extracts of the pomace can also be used.Extracts of skins can also be used or prepared.

In addition to the grape seed products disclosed herein other grapeproducts can also be used in the methods. The other grape seed productscan be used in the place of, or in combination with, the Chardonnay seedproducts. Examples of other grape seed products include, but are notlimited to, Syrah grape seed products, Cabernet Sauvignon grape seedproducts, Pinot Noir grape seed products, Sauvignon Blanc grape seedproducts, White Riesling grape seed products, and the like.

The seed products can be included in a variety of food products, such asnutritional beverages (e.g., nutritional shakes), baked goods (e.g.,cookies, brownies, cake, breads, biscuits, crackers), puddings,confections (i.e., candy), snack foods (e.g., pretzels), ice cream,frozen confections and novelties, or non-baked, extruded food productssuch as bars, including health or energy bars. The Chardonnay seedproduct can also be provided as a nutritional supplement, either intablet or capsule form, or as a powder for use as a nutritional food orbeverage additive.

In some embodiments, the seed product can be blended with other dry foodmaterials for use in the preparation of food products enriched with seedproducts. Dry food materials include, for example, dry starch-containingmaterials, dry protein-containing materials or combinations thereof.Suitable starch-containing materials may be derived from, for example,rice, corn, soybeans, hemp, sunflower, canola, wheat, oats, rye, potato,or any combination thereof. Suitable dry protein-containing materialsmay be derived from for example, meat, milk, fish or any combinationthereof. For baking applications, the seed product is suitable used inan amount ranging from 3% to 15% of the dry food material (e.g., whiteor whole wheat flour). The dry food may optionally also includeadditional ingredients such as vitamins, mineral fortifiers, salts,colors, flavors, flavor enhancers or sweeteners.

Seed products can be incorporated into beverages, processed meats,frozen desserts, confectionery products, dairy-type products, saucecompositions, and cereal grain products. Beverage products include, forexample, smoothies, infant formula, fruit juice beverages, yogurtbeverages, coffee beverages, beers, dry beverage mixes, tea fusionbeverages, sports beverages, soy liquors, casca seca, soda, slushes, andfrozen beverage mixes. Meat products include, for example, groundchicken products, water-added ham products, bologna, hot dogs, franks,chicken patties, chicken nuggets, beef patties, fish patties, surimi,bacon, luncheon meat, sandwich fillings, deli meats, meat snacks,meatballs, jerky, fajitas, bacon bits, injected meats, and bratwurst.Confectionery products include, for example, chocolates, mousses,chocolate coatings, yogurt coatings, cocoa, frostings, candies, energybars, and candy bars. Frozen dessert products include, for example, icecream, malts, shakes, popsicles, sorbets, and frozen pudding products.Dairy-type products include, for example, yogurt, cheese, ice cream,whipped topping, coffee creamer, cream cheese, sour cream, cottagecheese, butter, mayonnaise, milk-based sauces, milk-based saladdressings, and cheese curds. Cereal grain products include, for example,breads, muffins, bagels, pastries, noodles, cookies, pancakes, waffles,biscuits, semolina, chips, tortillas, cakes, crackers, breakfast cereals(including both ready-to-eat and cooked cereals), pretzels, dry bakerymixes, melba toast, breadsticks, croutons, stuffing, energy bars,doughnuts, cakes, popcorn, taco shells, fry coatings, batters, breading,crusts, brownies, pies, puffed soy cakes, crepes, croissants, flour, andpolenta. Sauce compositions include salad dressings, nut butter spreads(e.g., peanut butter spreads), marinades, sauces, salsas, jams, cheesesauces, mayonnaise, tartar sauce, soy humus, dips, fruit syrups, andmaple syrups. Sauce composition may also include a suspending agent toaid in maintaining the uniformity of the composition. Examples ofsuitable suspending agents include polysaccharides, such as starch,cellulose (e.g., microcrystalline cellulose) and carrageenan, andpolyuronides, such as pectin. Gelatin is another example of a suspendingagent which may be used in the beverage compositions as well. Examplesof additional supplemented food products prepared using the premixesdescribed herein include, but are not limited to, tofu, formulated soyessence, powdered protein supplements, juice mixable proteinsupplements, foaming agents, clouding agents, baby foods, meatlessballs, meat analogues, egg products (e.g., scrambled eggs), soups,chowders, broth, milk alternatives, soy-milk products, chili, spicemixes, sprinkles, soy whiz, salad topping, edible films, edible sticks,chewing gum, bacon bits, veggie bits, pizza crust barriers, soy pie,no-gas synthetic beans, soy helper, soy cotton candy, fruit bits, pizzarolls, mashed potatoes, spun soy protein fiber, soy roll-ups, extrudedsnacks, condiments, lotions, fries, gelatin dessert products, vitaminsupplements, nutritional bars, dry cake, bread or muffin mixes, andmicrowavable instant dry mixes.

In some embodiments, the seed product may be provided as an energy bar(suitable for consumption during physical activity) or a mealreplacement bar. The energy bar or meal replacement bar can also containone or more vitamin, mineral, food supplement, botanical, or plant orherb extracts or ingredients known in the art or used in energy bars ormeal replacement bars, such as a fruit juice or extract, an herb or herbflavor, natural or artificial flavors, vitamins, minerals, anti-oxidantcontaining extracts, coenzyme Q, omega-3 fatty acids, guarana, caffeine,theobromine, maltodextrin, and protein. In some embodiments, the energybar or meal replacement bar can have total available energy levels ofcarbohydrates/protein/fat of 40/30/30 respectively.

The energy and meal replacement bars can be further supplemented forathletic performance enhancement, mental energy or cognitive focusenhancement, and/or nutritional benefit. Exemplary supplements include,but are not limited to Vinpocetine, Vincamine Ginkgo Biloba, L-Arginine,Acetyl-L-Carnitine, Feverfew, DMAE (Dimethylaminoethanol), DMAEbitartrate, P-chlorophenoxyacetate, Vitamin B-Complex, Ginseng, 5 HTP(5-Hydroxytryptophan), L-Theanine, Androstenedione, L-Glutamine,L-Tyrosine, L-Glycine, L-lysine, Whey Protein, and DHEA(Dehydroepiandrosterone), or any combination thereof.

The seed product or composition can also contain an amount of one ormore additional grape seeds or grape skin products which is/are not thefirst seed product. In some embodiments, an amount of the first seedproduct is replaced in the seed composition with an amount of the secondgrape seed or grape skin product. The amount of second grape seed orgrape skin product that will need to be added to the seed composition toattain the same benefit as a given amount of seed product can readily bedetermined by those skilled in the art.

The seed products prepared according to the methods described herein canalso be used, for example, in pharmaceutical compositions andformulations. The pharmaceutical compositions can be formulated bystandard techniques using one or more physiologically acceptablecarriers or excipients. In some embodiments, the formulations maycontain a buffer and/or a preservative. The compounds and theirphysiologically acceptable salts and solvates can be formulated foradministration by any suitable route, including via inhalation,topically, nasally, orally, parenterally (e.g., intravenously,intraperitoneally, intravesically or intrathecally) or rectally in avehicle comprising one or more pharmaceutically acceptable carriers, theproportion of which is determined by the solubility and chemical natureof the peptide, chosen route of administration and standard biologicalpractice.

According to some embodiments, pharmaceutical compositions are providedcomprising effective amounts of one or more compound(s) made accordingto the methods described herein together with, for example,pharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or other carriers. Such compositions includediluents of various buffer content (e.g., TRIS or other amines,carbonates, phosphates, amino acids, for example, glycinamidehydrochloride (especially in the physiological pH range),N-glycylglycine, sodium or potassium phosphate (dibasic, tribasic), etc.or TRIS-HCl or acetate), pH and ionic strength; additives such asdetergents and solubilizing agents (e.g., surfactants such as Pluronics,Tween 20, Tween 80 (Polysorbate 80), Cremophor, polyols such aspolyethylene glycol, propylene glycol, etc.), anti-oxidants (e.g.,ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol,benzyl alcohol, parabens, etc.) and bulking substances (e.g., sugarssuch as sucrose, lactose, mannitol, polymers such aspolyvinylpyrrolidones or dextran, etc.); and/or incorporation of thematerial into particulate preparations of polymeric compounds such aspolylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronicacid may also be used. Such compositions can be employed to influencethe physical state, stability, rate of in vivo release, and rate of invivo clearance of a compound of the present invention. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated byreference. The compositions can, for example, be prepared in liquidform, or can be in dried powder, such as lyophilized form. Particularmethods of administering such compositions are described infra. Thecompositions can also be crystallized or precipitated. Examples ofmethods of crystallizing or precipitating can be found, for example, inU.S. Provisional Application No. 61/936,914, filed Feb. 7, 2014, whichis hereby incorporated by reference in its entirety.

Where a buffer is to be included in the formulations, the buffer can beselected from the group consisting of sodium acetate, sodium carbonate,citrate, glycylglycine, histidine, glycine, lysine, arginin, sodiumdihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, or mixtures thereof. Each one of thesespecific buffers constitutes an alternative embodiment. In someembodiments the buffer is glycylglycine, sodium dihydrogen phosphate,disodium hydrogen phosphate, sodium phosphate or mixtures thereof.

Where a pharmaceutically acceptable preservative is to be included inthe formulations of the invention, the preservative is selected from thegroup consisting of phenol, m-cresol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, ormixtures thereof. Each one of these specific preservatives constitutesan alternative embodiment. In some embodiments, the preservative isphenol or m-cresol.

In some embodiments, the preservative is present in a concentration fromabout 0.1 mg/ml to about 50 mg/ml, in a concentration from about 0.1mg/ml to about 25 mg/ml, or in a concentration from about 0.1 mg/ml toabout 10 mg/ml. In some embodiments, the preservative is present in aconcentration from about 1 mg/ml to about 50 mg/ml, in a concentrationfrom about 1 mg/ml to about 25 mg/ml, or in a concentration from about 1mg/ml to about 10 mg/ml.

The use of a preservative in pharmaceutical compositions is well-knownto the skilled person. For convenience reference is made to Remington:The Science and Practice of Pharmacy, 19th edition, 1995.

In some embodiments, the formulation may further comprise a chelatingagent where the chelating agent may be selected from salts ofethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid,and mixtures thereof. Each one of these specific chelating agentsconstitutes an alternative embodiment.

In some embodiments, the chelating agent is present in a concentrationfrom 0.1 mg/ml to 5 mg/ml, from 0.1 mg/ml to 2 mg/ml, or from 2 mg/ml to5 mg/ml.

The use of a chelating agent in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 19th edition, 1995.

In some embodiments, the formulation may further comprise a stabilizerselected from the group of high molecular weight polymers or lowmolecular compounds where such stabilizers include, but are not limitedto, polyethylene glycol (e.g. PEG 3350), polyvinylalcohol (PVA),polyvinylpyrrolidone, carboxymethylcellulose, different salts (e.g.sodium chloride), L-glycine, L-histidine, imidazole, arginine, lysine,isoleucine, aspartic acid, tryptophan, threonine and mixtures thereof.Each one of these specific stabilizers constitutes an alternativeembodiment. In some embodiments, the stabilizer is selected from thegroup consisting of L-histidine, imidazole and arginine.

In some embodiments, the high molecular weight polymer is present in aconcentration from 0.1 mg/ml to 50 mg/ml, from 0.1 mg/ml to 5 mg/ml,from 5 mg/ml to 10 mg/ml, from 10 mg/ml to 20 mg/ml, from 20 mg/ml to 30mg/ml or from 30 mg/ml to 50 mg/ml.

In some embodiments, the low molecular weight compound is present in aconcentration from 0.1 mg/ml to 50 mg/ml, from 0.1 mg/ml to 5 mg/ml,from 5 mg/ml to 10 mg/ml, from 10 mg/ml to 20 mg/ml, from 20 mg/ml to 30mg/ml, or from 30 mg/ml to 50 mg/ml.

The use of a stabilizer in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19th edition, 1995.

In some embodiments, the formulation may further comprise a surfactantwhere a surfactant may be selected from a detergent, ethoxylated castoroil, polyglycolyzed glycerides, acetylated monoglycerides, sorbitanfatty acid esters, poloxamers, such as 188 and 407, polyoxyethylenesorbitan fatty acid esters, polyoxyethylene derivatives such asalkylated and alkoxylated derivatives (tweens, e.g. Tween-20, orTween-80), monoglycerides or ethoxylated derivatives thereof,diglycerides or polyoxyethylene derivatives thereof, glycerol, cholicacid or derivatives thereof, lecithins, alcohols and phospholipids,glycerophospholipids (lecithins, kephalins, phosphatidyl serine),glyceroglycolipids (galactopyransoide), sphingophospholipids(sphingomyelin), and sphingoglycolipids (ceramides, gangliosides), DSS(docusate sodium, docusate calcium, docusate potassium, SDS (sodiumdodecyl sulfate or sodium lauryl sulfate), dipalmitoyl phosphatidicacid, sodium caprylate, bile acids and salts thereof and glycine ortaurine conjugates, ursodeoxycholic acid, sodium cholate, sodiumdeoxycholate, sodium taurocholate, sodium glycocholate,N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic(alkyl-aryl-sulphonates) monovalent surfactants, palmitoyllysophosphatidyl-L-serine, lysophospholipids (e.g.1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine), alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the postively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine,zwitterionic surfactants (e.g.N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate,dodecylphosphocholine, myristoyl lysophosphatidylcholine, hen egglysolecithin), cationic surfactants (quarternary ammonium bases) (e.g.cetyl-trimethylammonium bromide, cetylpyridinium chloride), non-ionicsurfactants, polyethyleneoxide/polypropyleneoxide block copolymers(Pluronics/Tetronics, Triton X-100, Dodecyl β-D-glucopyranoside) orpolymeric surfactants (Tween-40, Tween-80, Brij-35), fusidic acidderivatives—(e.g. sodium tauro-dihydrofusidate etc.), long-chain fattyacids and salts thereof C6-C12 (e.g. oleic acid and caprylic acid),acylcarnitines and derivatives, Nα-acylated derivatives of lysine,arginine or histidine, or side-chain acylated derivatives of lysine orarginine, Nα-acylated derivatives of dipeptides comprising anycombination of lysine, arginine or histidine and a neutral or acidicamino acid, Nα-acylated derivative of a tripeptide comprising anycombination of a neutral amino acid and two charged amino acids, or thesurfactant may be selected from the group of imidazoline derivatives, ormixtures thereof. Each one of these specific surfactants constitutes analternative embodiment.

The use of a surfactant in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19th edition, 1995.

In some embodiments, the composition can comprises a sweetener,including pharmaceutically acceptable sweeteners. Examples ofpharmaceutically acceptable sweeteners can include at least one intensesweetener such as saccharin, sodium or calcium saccharin, aspartame,acesulfame potassium, sodium cyclamate, alitame, a dihydrochalconesweetener, monellin, stevioside or sucralose(4,1′,6′-trichloro-4,1′,6′-trideoxygalactosucrose). In some embodiments,it includes saccharin, sodium or calcium saccharin, and optionally abulk sweetener such as sorbitol, mannitol, fructose, sucrose, maltose,isomalt, glucose, hydrogenated glucose syrup, xylitol, caramel or honey.

Intense sweeteners can also be used, for example, in low concentrations.For example, in the case of sodium saccharin, the concentration mayrange from 0.04% to 0.1% (w/v) based on the total volume of the finalformulation, and can be about 0.06% in the low-dosage formulations andabout 0.08% in the high-dosage ones. The bulk sweetener can effectivelybe used in larger quantities ranging from about 10% to about 35% or fromabout 10% to 15% (w/v).

The formulations may be prepared by conventional techniques, e.g. asdescribed in Remington's Pharmaceutical Sciences, 1985 or in Remington:The Science and Practice of Pharmacy, 19th edition, 1995, where suchconventional techniques of the pharmaceutical industry involvedissolving and mixing the ingredients as appropriate to give the desiredend product.

The compositions and products described herein can also be administeredby any method known in the art. For example, administration may betransdermal, parenteral, intravenous, intra-arterial, subcutaneous,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intracapsular, intraspinal, intracisternal, intraperitoneal,intracerebroventricular, intrathecal, intranasal, aerosol, bysuppositories, or oral administration. In some embodiments, apharmaceutical composition of the compounds prepared herein can be foradministration for injection, or for oral, pulmonary, nasal,transdermal, ocular administration.

For oral administration, the composition can be formulated in unitdosage forms such as capsules or tablets. The tablets or capsules may beprepared by conventional methods with pharmaceutically acceptableexcipients, including binding agents, for example, pregelatinised maizestarch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose; fillers,for example, lactose, microcrystalline cellulose, or calcium hydrogenphosphate; lubricants, for example, magnesium stearate, talc, or silica;disintegrants, for example, potato starch or sodium starch glycolate; orwetting agents, for example, sodium lauryl sulphate. Tablets can becoated by methods well known in the art. Liquid preparations for oraladministration can take the form of, for example, solutions, syrups, orsuspensions, or they can be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations can be prepared by conventional means with pharmaceuticallyacceptable additives, for example, suspending agents, for example,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats;emulsifying agents, for example, lecithin or acacia; non-aqueousvehicles, for example, almond oil, oily esters, ethyl alcohol, orfractionated vegetable oils; and preservatives, for example, methyl orpropyl-p-hydroxybenzoates or sorbic acid. The preparations can alsocontain buffer salts, flavoring, coloring, and/or sweetening agents asappropriate. If desired, preparations for oral administration can besuitably formulated to give controlled release of the active compound.The compounds can also be prepared with cyclodextrins or other largemolecules to facilitate oral adsorption.

For topical administration, the composition can be formulated in apharmaceutically acceptable vehicle containing 0.1 to 10 percent or 0.5to 5 percent of the active compound(s). Such formulations can be in theform of a cream, lotion, sublingual tablet, aerosols and/or emulsionsand can be included in a transdermal or buccal patch of the matrix orreservoir type as are conventional in the art for this purpose.

For parenteral administration, the compositions can be administered byintravenous, subcutaneous, or intramuscular injection, in compositionswith pharmaceutically acceptable vehicles or carriers. The compositionscan be formulated for parenteral administration by injection, forexample, by bolus injection or continuous infusion. Formulations forinjection can be presented in unit dosage form, for example, in ampoulesor in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and can contain formulatory agents, forexample, suspending, stabilizing, and/or dispersing agents.Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use.

For administration by injection, the compositions can be administered insolution in a sterile aqueous vehicle which may also contain othersolutes such as buffers or preservatives as well as sufficientquantities of pharmaceutically acceptable salts or of glucose to makethe solution isotonic. In some embodiments, the pharmaceuticalcompositions may be formulated with a pharmaceutically acceptablecarrier to provide sterile solutions or suspensions for injectableadministration. In particular, injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspensions in liquid prior to injectionor as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, or the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations(e.g., liposomes) may be utilized. Suitable pharmaceutical carriers aredescribed in “Remington's pharmaceutical Sciences” by E. W. Martin.

For administration by inhalation, the compositions may be convenientlydelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, forexample, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In thecase of a pressurized aerosol, the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator can beformulated containing a powder mix of the compound and a suitable powderbase, for example, lactose or starch. For intranasal administration thecompositions of the invention may be used, for example, as a liquidspray, as a powder or in the form of drops.

The compositions can also be formulated in rectal compositions, forexample, suppositories or retention enemas, for example, containingconventional suppository bases, for example, cocoa butter or otherglycerides.

Furthermore, the compositions can be formulated as a depot preparation.Such long-acting formulations can be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds can be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can, for example, comprise metal or plasticfoil, for example, a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

The compositions prepared according to the methods described herein maybe administered to a patient at effective doses, includingtherapeutically effective doses, to prevent, treat, or control diseasesand disorders described herein. They can also be used to restore ormaintain a healthy liver as described herein. Pharmaceuticalcompositions may be administered to a patient in an amount sufficient toelicit an effective maintenance, protective, restorative, or therapeuticresponse in the patient. An amount adequate to accomplish this isdefined as “therapeutically effective dose.” The dose will be determinedby the efficacy of the particular composition employed and the conditionof the subject, as well as the body weight or surface area of the areato be treated. The size of the dose also will be determined by theexistence, nature, and extent of any adverse effects that accompany theadministration of a particular compound or vector in a particularsubject.

Toxicity and therapeutic efficacy of such compositions can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, for example, by determining the LD50 (the dose lethal to 50% ofthe population) and the ED50 (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and can be expressed as the ratio, LD50/ED50.Compositions that exhibit large therapeutic indices are preferred. Whilecompositions that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue to minimize potential damage to normal cellsand, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can beused to formulate a dosage range for use in humans. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration. For any composition used in the methods describedherein, the therapeutically effective dose can be estimated initially,for example, from cell culture assays. A dose can be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC50 (the concentration of the test compound that achievesa half-maximal inhibition of symptoms) as determined in cell culture.Such information can be used to more accurately determine useful dosesin humans. Levels in plasma can be measured, for example, by highperformance liquid chromatography (HPLC). In general, the doseequivalent of a modulator is from about 1 ng/kg to 10 mg/kg for atypical subject or as described elsewhere herein.

The amount and frequency of administration of the compositions will beregulated according to the judgment of the user or attending clinicianconsidering such factors as age, condition and size of the patient aswell as severity of the symptoms being treated. An ordinarily skilledphysician or veterinarian can readily determine and prescribe theeffective amount of the drug required to prevent, counter or arrest theprogress of the condition. In general it is contemplated that aneffective amount would be from 0.001 mg/kg to 10 mg/kg body weight, andin particular from 0.01 mg/kg to 1 mg/kg body weight or as otherwisedescribed herein. In some embodiments, it is contemplated that aneffective amount would be to continuously infuse by intravenousadministration from 0.01 micrograms/kg body weight/min to 100micrograms/kg body weight/min for a period of 12 hours to 14 days. Itmay be appropriate to administer the required dose as two, three, fouror more sub-doses at appropriate intervals throughout the day. Saidsub-doses may be formulated as unit dosage forms, for example,containing 0.01 to 500 mg, and in particular 0.1 mg to 200 mg of thecomposition per unit dosage form.

In some embodiments, the pharmaceutical preparation is in a unit dosageform. In such form, the preparation is subdivided into suitably sizedunit doses containing appropriate quantities of the active component,e.g., an effective amount to achieve the desired purpose. The quantityof active compound in a unit dose of preparation may be varied oradjusted from about 0.01 mg to about 1000 mg, from about 0.01 mg toabout 750 mg, from about 0.01 mg to about 500 mg, or from about 0.01 mgto about 250 mg, according to the particular application. The actualdosage employed may be varied depending upon the requirements of thepatient and the severity of the condition being treated. Determinationof the proper dosage regimen for a particular situation is within theskill of the art. For convenience, the total dosage may be divided andadministered in portions during the day as required.

In some embodiment of the methods, the amount of grape product consumedas a percentage of daily diet is at least 3%, at least 5%, or at least8% by mass. In some embodiments, 5-10%, 7%, or 10% of the daily diet bymass is consumed.

In some embodiments of the methods, the amount of grape product consumedas a percentage of daily diet is at least 3%, at least 5%, or at least8% of total calories consumed. In some embodiments, 5-10%, 7%, or 10% ofthe daily calories are from the grape product.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 1 mg to about 15,000 mg, from about 1 mg to about14,000 mg, about 1 mg to about 13,000 mg, about 1 mg to about 12,000 mg,about 1 mg to about 11,000 mg, about 1 mg to about 10,000 mg, about 1 mgto about 9,000 mg, about 1 mg to about 8,000 mg, about 1 mg to about7,000 mg, about 1 mg to about 6,000 mg, about 1 mg to about 5,000 mg,about 1 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 1 mgto about 2,000 mg, about 1 mg to about 1,000 mg, about 1 mg to about 900mg, about 1 mg to about 800 mg, about 1 mg to about 700 mg, about 1 mgto about 600 mg, about 1 mg to about 500 mg, about 1 mg to about 400 mg,about 1 mg to about 300 mg, about 1 mg to about 200 mg, about 1 mg toabout 100 mg, about 1 mg to about 75 mg, about 1 mg to about 50 mg, orabout 1 mg to about 25 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 5 mg to about 15,000 mg, from about 5 mg to about14,000 mg, about 5 mg to about 13,000 mg, about 5 mg to about 12,000 mg,about 5 mg to about 11,000 mg, about 5 mg to about 10,000 mg, about 5 mgto about 9,000 mg, about 5 mg to about 8,000 mg, about 5 mg to about7,000 mg, about 5 mg to about 6,000 mg, about 5 mg to about 5,000 mg,about 5 mg to about 4,000 mg, about 5 mg to about 3,000 mg, about 5 mgto about 2,000 mg, about 5 mg to about 1,000 mg, about 5 mg to about 900mg, about 5 mg to about 800 mg, about 5 mg to about 700 mg, about 5 mgto about 600 mg, about 5 mg to about 500 mg, about 5 mg to about 400 mg,about 5 mg to about 300 mg, about 5 mg to about 200 mg, about 5 mg toabout 100 mg, about 5 mg to about 75 mg, about 5 mg to about 50 mg, orabout 5 mg to about 25 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 10 mg to about 15,000 mg, from about 10 mg to about14,000 mg, about 10 mg to about 13,000 mg, about 10 mg to about 12,000mg, about 10 mg to about 11,000 mg, about 10 mg to about 10,000 mg,about 10 mg to about 9,000 mg, about 10 mg to about 8,000 mg, about 10mg to about 7,000 mg, about 10 mg to about 6,000 mg, about 10 mg toabout 5,000 mg, about 10 mg to about 4,000 mg, about 10 mg to about3,000 mg, about 10 mg to about 2,000 mg, about 10 mg to about 1,000 mg,about 10 mg to about 900 mg, about 10 mg to about 800 mg, about 10 mg toabout 700 mg, about 10 mg to about 600 mg, about 10 mg to about 500 mg,about 10 mg to about 400 mg, about 10 mg to about 300 mg, about 10 mg toabout 200 mg, about 10 mg to about 100 mg, about 10 mg to about 75 mg,about 10 mg to about 50 mg, or about 10 mg to about 25 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 25 mg to about 15,000 mg, from about 25 mg to about14,000 mg, about 25 mg to about 13,000 mg, about 25 mg to about 12,000mg, about 25 mg to about 11,000 mg, about 25 mg to about 10,000 mg,about 25 mg to about 9,000 mg, about 25 mg to about 8,000 mg, about 25mg to about 7,000 mg, about 25 mg to about 6,000 mg, about 25 mg toabout 5,000 mg, about 25 mg to about 4,000 mg, about 25 mg to about3,000 mg, about 25 mg to about 2,000 mg, about 25 mg to about 1,000 mg,about 25 mg to about 900 mg, about 25 mg to about 800 mg, about 25 mg toabout 700 mg, about 25 mg to about 600 mg, about 25 mg to about 500 mg,about 25 mg to about 400 mg, about 25 mg to about 300 mg, about 25 mg toabout 200 mg, about 25 mg to about 100 mg, about 25 mg to about 75 mg,or about 25 mg to about 50 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 50 mg to about 15,000 mg, from about 50 mg to about14,000 mg, about 50 mg to about 13,000 mg, about 50 mg to about 12,000mg, about 50 mg to about 11,000 mg, about 50 mg to about 10,000 mg,about 50 mg to about 9,000 mg, about 50 mg to about 8,000 mg, about 50mg to about 7,000 mg, about 50 mg to about 6,000 mg, about 50 mg toabout 5,000 mg, about 50 mg to about 4,000 mg, about 50 mg to about3,000 mg, about 50 mg to about 2,000 mg, about 50 mg to about 1,000 mg,about 50 mg to about 900 mg, about 50 mg to about 800 mg, about 50 mg toabout 700 mg, about 50 mg to about 600 mg, about 50 mg to about 500 mg,about 50 mg to about 400 mg, about 50 mg to about 300 mg, about 50 mg toabout 200 mg, about 50 mg to about 100 mg, or about 50 mg to about 75mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 75 mg to about 15,000 mg, from about 75 mg to about14,000 mg, about 75 mg to about 13,000 mg, about 75 mg to about 12,000mg, about 75 mg to about 11,000 mg, about 75 mg to about 10,000 mg,about 75 mg to about 9,000 mg, about 75 mg to about 8,000 mg, about 75mg to about 7,000 mg, about 75 mg to about 6,000 mg, about 75 mg toabout 5,000 mg, about 75 mg to about 4,000 mg, about 75 mg to about3,000 mg, about 75 mg to about 2,000 mg, about 75 mg to about 1,000 mg,about 75 mg to about 900 mg, about 75 mg to about 800 mg, about 75 mg toabout 700 mg, about 75 mg to about 600 mg, about 75 mg to about 500 mg,about 75 mg to about 400 mg, about 75 mg to about 300 mg, about 75 mg toabout 200 mg, or about 75 mg to about 100 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 100 mg to about 15,000 mg, from about 100 mg toabout 14,000 mg, about 100 mg to about 13,000 mg, about 100 mg to about12,000 mg, about 100 mg to about 11,000 mg, about 100 mg to about 10,000mg, about 100 mg to about 9,000 mg, about 100 mg to about 8,000 mg,about 100 mg to about 7,000 mg, about 100 mg to about 6,000 mg, about100 mg to about 5,000 mg, about 100 mg to about 4,000 mg, about 100 mgto about 3,000 mg, about 100 mg to about 2,000 mg, about 100 mg to about1,000 mg, about 100 mg to about 900 mg, about 100 mg to about 800 mg,about 100 mg to about 700 mg, about 100 mg to about 600 mg, about 100 mgto about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 300mg, or about 100 mg to about 200 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 200 mg to about 15,000 mg, from about 200 mg toabout 14,000 mg, about 200 mg to about 13,000 mg, about 200 mg to about12,000 mg, about 200 mg to about 11,000 mg, about 200 mg to about 10,000mg, about 200 mg to about 9,000 mg, about 200 mg to about 8,000 mg,about 200 mg to about 7,000 mg, about 200 mg to about 6,000 mg, about200 mg to about 5,000 mg, about 200 mg to about 4,000 mg, about 200 mgto about 3,000 mg, about 200 mg to about 2,000 mg, about 200 mg to about1,000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg,about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 200 mgto about 500 mg, about 200 mg to about 400 mg, or about 200 mg to about300 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 300 mg to about 15,000 mg, from about 300 mg toabout 14,000 mg, about 300 mg to about 13,000 mg, about 300 mg to about12,000 mg, about 300 mg to about 11,000 mg, about 300 mg to about 10,000mg, about 300 mg to about 9,000 mg, about 300 mg to about 8,000 mg,about 300 mg to about 7,000 mg, about 300 mg to about 6,000 mg, about300 mg to about 5,000 mg, about 300 mg to about 4,000 mg, about 300 mgto about 3,000 mg, about 300 mg to about 2,000 mg, about 300 mg to about1,000 mg, about 300 mg to about 900 mg, about 300 mg to about 800 mg,about 300 mg to about 700 mg, about 300 mg to about 600 mg, about 300 mgto about 500 mg, or about 300 mg to about 400 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 400 mg to about 15,000 mg, from about 400 mg toabout 14,000 mg, about 400 mg to about 13,000 mg, about 400 mg to about12,000 mg, about 400 mg to about 11,000 mg, about 400 mg to about 10,000mg, about 400 mg to about 9,000 mg, about 400 mg to about 8,000 mg,about 400 mg to about 7,000 mg, about 400 mg to about 6,000 mg, about400 mg to about 5,000 mg, about 400 mg to about 4,000 mg, about 400 mgto about 3,000 mg, about 400 mg to about 2,000 mg, about 400 mg to about1,000 mg, about 400 mg to about 900 mg, about 400 mg to about 800 mg,about 400 mg to about 700 mg, about 400 mg to about 600 mg, about 400 mgto about 500 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 500 mg to about 15,000 mg, from about 500 mg toabout 14,000 mg, about 500 mg to about 13,000 mg, about 500 mg to about12,000 mg, about 500 mg to about 11,000 mg, about 500 mg to about 10,000mg, about 500 mg to about 9,000 mg, about 500 mg to about 8,000 mg,about 500 mg to about 7,000 mg, about 500 mg to about 6,000 mg, about500 mg to about 5,000 mg, about 500 mg to about 4,000 mg, about 500 mgto about 3,000 mg, about 500 mg to about 2,000 mg, about 500 mg to about1,000 mg, about 500 mg to about 900 mg, about 500 mg to about 800 mg,about 500 mg to about 700 mg, or about 500 mg to about 600 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 600 mg to about 15,000 mg, from about 600 mg toabout 14,000 mg, about 600 mg to about 13,000 mg, about 600 mg to about12,000 mg, about 600 mg to about 11,000 mg, about 600 mg to about 10,000mg, about 600 mg to about 9,000 mg, about 600 mg to about 8,000 mg,about 600 mg to about 7,000 mg, about 600 mg to about 6,000 mg, about600 mg to about 5,000 mg, about 600 mg to about 4,000 mg, about 600 mgto about 3,000 mg, about 600 mg to about 2,000 mg, about 600 mg to about1,000 mg, about 600 mg to about 900 mg, about 600 mg to about 800 mg, orabout 600 mg to about 700 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 700 mg to about 15,000 mg, from about 700 mg toabout 14,000 mg, about 700 mg to about 13,000 mg, about 700 mg to about12,000 mg, about 700 mg to about 11,000 mg, about 700 mg to about 10,000mg, about 700 mg to about 9,000 mg, about 700 mg to about 8,000 mg,about 700 mg to about 7,000 mg, about 700 mg to about 6,000 mg, about700 mg to about 5,000 mg, about 700 mg to about 4,000 mg, about 700 mgto about 3,000 mg, about 700 mg to about 2,000 mg, about 700 mg to about1,000 mg, about 700 mg to about 900 mg, or about 700 mg to about 800 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 800 mg to about 15,000 mg, from about 800 mg toabout 14,000 mg, about 800 mg to about 13,000 mg, about 800 mg to about12,000 mg, about 800 mg to about 11,000 mg, about 800 mg to about 10,000mg, about 800 mg to about 9,000 mg, about 800 mg to about 8,000 mg,about 800 mg to about 7,000 mg, about 800 mg to about 6,000 mg, about800 mg to about 5,000 mg, about 800 mg to about 4,000 mg, about 800 mgto about 3,000 mg, about 800 mg to about 2,000 mg, about 800 mg to about1,000 mg, or about 800 mg to about 900 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 900 mg to about 15,000 mg, from about 900 mg toabout 14,000 mg, about 900 mg to about 13,000 mg, about 900 mg to about12,000 mg, about 900 mg to about 11,000 mg, about 900 mg to about 10,000mg, about 900 mg to about 9,000 mg, about 900 mg to about 8,000 mg,about 900 mg to about 7,000 mg, about 900 mg to about 6,000 mg, about900 mg to about 5,000 mg, about 900 mg to about 4,000 mg, about 900 mgto about 3,000 mg, about 900 mg to about 2,000 mg, or about 900 mg toabout 1,000 mg.

In some embodiments of the methods, the grape product, whole pomace orany portion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing is administered inan amount from about 1,000 mg to about 15,000 mg, from about 1,000 mg toabout 14,000 mg, about 1,000 mg to about 13,000 mg, about 1,000 mg toabout 12,000 mg, about 1,000 mg to about 11,000 mg, about 1,000 mg toabout 10,000 mg, about 1,000 mg to about 9,000 mg, about 1,000 mg toabout 8,000 mg, about 1,000 mg to about 7,000 mg, about 1,000 mg toabout 6,000 mg, about 1,000 mg to about 5,000 mg, about 1,000 mg toabout 4,000 mg, about 1,000 mg to about 3,000 mg, or about 1,000 mg toabout 2,000 mg.

In some embodiments, the amount of grape product, whole pomace or anyportion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing consumed daily isat least, or about 0.1 g, at least, or about, 0.5 g, is at least, orabout, 1 g, is at least, or about, 2 g, is at least, or about, 3 g, isat least, or about, 4 g, is at least, or about 5 g, at least, or about,10 g, at least, or about, 15 g, at least, or about, 20 g, at least, orabout, 25 g, at least, or about, 30 g, at least, or about, 35 g, atleast, or about, 40 g, or at least, or about, 45 g. In some embodiments,at least, or about, 50 g of the grape product is consumed daily.

In some embodiments, the amount of grape product, whole pomace or anyportion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing consumed daily isat least 1 tablespoon, at least 2 tablespoons, at least 3 tablespoons,at least 4 tablespoons, or at least 5 tablespoons.

In some embodiments, the amount of grape product, whole pomace or anyportion thereof, skin product, seed flour, or seed product, or anyextract thereof, or any combination of the foregoing consumed daily on aweight:body weight basis is at least 0.2 g/kg, at least 0.5 g/kg, or atleast 0.7 g/kg. In some embodiments, at least 1 g of the grape productper kg of body weight is consumed per day.

Seed flour can be substituted or combined with whole pomace or anyportion thereof, pomace meal, pomace flour, skin flour, skin product,seed product, or seed meal, or any extract of the thereof, or anycombination of the foregoing is administered in any of the methods orembodiments disclosed herein. The amount of pomace meal, pomace flour,skin flour, seed extract, or seed meal that will need to be consumeddaily to attain the same benefit as a given amount of seed flour canreadily be determined by those skilled in the art and as describedherein. For example, it is expected that a subject will need to consumeabout three times as much skin flour to achieve the same benefit as agiven amount of seed flour.

In some embodiments, the grape product is taken at least twice a week,at least 3 times a week, or every other day. In some embodiments, thegrape product is incorporated into the daily diet. In some embodiments,the product is administered twice a day, three times a day, or fourtimes a day.

The grape product can be taken for an amount of time sufficient to treatand/or prevent a condition amenable to treatment and/or prevention byseed product as described herein. The grape product can be taken for atleast one week, at least 2 weeks, at least 3 weeks, at least one month,at least 2 months, at least 3 months, at least 6 months, at least ayear, or indefinitely.

In some embodiments of the methods, a second grape product which is notthe first grape product is administered to the individual. In someembodiments, the combination of the first grape product and second grapeproduct (seed, skin, pomace, and the like) provides a therapeutic effector health benefit which is greater than the effect of administration offirst grape product alone.

In some embodiments, the amount of the first grape product and amount ofthe second grape product or grape seed, pomace, or skin product areselected so that the effect achieved is at least the same as the effectachieved by a given amount of the first seed product administered alone.

In some embodiments, methods of treating or preventing non-alcoholicfatty liver disease (NAFLD) in a mammal are provided. In someembodiments, the methods comprise administering to the mammal an amountof grape product effective to treat or prevent NAFLD. In someembodiments, the grape product is a grape seed product. In someembodiments, the grape product is a combination or extract of thedifferent grape products described herein. In some embodiments, the seedproduct is Chardonnay seed product. Nonalcoholic fatty liver disease isa term used to describe the accumulation of fat in the liver of peoplewho drink little or no alcohol. In some embodiments, mammal consumes noalcohol. In some embodiments, the mammal has inflammation of the liver.In some embodiments, the mammal has scarring in the liver. The form ofNAFLD with the scarring can be referred to as nonalcoholicsteatohepatitis. In some embodiments, the mammal with NAFLD can alsohave liver failure. The compositions described herein can be used totreat or ameliorate any of these conditions.

In some embodiments, methods of reducing hepatic steatosis in a mammalcomprising administering to the mammal an amount of a grape producteffective to reduce hepatic steatosis. In some embodiments, the grapeproduct is a Chardonnay grape product.

In some embodiments, methods of reducing steatohepatitis in a mammalcomprising administering to the mammal an amount of a grape producteffective to reduce steatohepatitis are provided. In some embodiments,the grape product is a Chardonnay grape product. In some embodiments,the grape product is a grape seed product or extract thereof.

In some embodiments, methods of reducing hepatic fibrosis in a mammalare provided. In some embodiments, the methods comprise administering tothe mammal an amount of a grape seed product effective to reduce hepaticfibrosis. In some embodiments, the grape product is a Chardonnay grapeproduct. In some embodiments, the grape product is a grape seed productor extract thereof.

In some embodiments, methods of treating or preventing non-alcoholicsteatohepatitis (NASH) in a mammal are provided. In some embodiments,the methods comprise administering to the mammal an amount of a grapeproduct effective to treat or prevent NASH. In some embodiments, thegrape product is a Chardonnay grape product. In some embodiments, thegrape product is a grape seed product or extract thereof.

In some embodiments, methods of supporting a healthy liver in a mammalare provided. In some embodiments, the method comprises administering tothe mammal an amount of a grape product effective to support a healthyliver. In some embodiments, the healthy liver is supported by preservingthe normal function of the liver. This can be done, for example, by theenhancing or preserving the liver's ability to metabolize energy. Insome embodiments, this is done by supporting the immune response toprotect and/or support the liver from external insult. In someembodiments, methods of controlling acute stress-induced damage to aliver in a subject due to exposure to environmental toxins and/orconsumed products are provided. In some embodiments, the methodcomprises administering to the subject an amount of a grape product asdescribed herein.

In some embodiments, methods of modulating miRNAs in a subject's liverthat ameliorate the toxic stress response is provided. In someembodiments, the method comprises administering to the subject an amountof a grape product as described herein.

In some embodiments, methods of increasing the ability of a liver in asubject to regenerate and/or heal are provided. In some embodiments, themethod comprises administering to the subject an amount of a grapeproduct as described herein.

The grape products can also be used to inhibit or treat oxidativestress. The products can also be used to maintain a healthy oxidativestress level in the mammal. The methods comprise administering to themammal an effective amount of a grape seed product described herein. Insome embodiments, the oxidative stress is present in the liver. In someembodiments, the grape seed products can be used to reduce liverinflammation.

In some embodiments, methods of reducing or preventing oxidative stressin a mammal's liver are provided. In some embodiments, the methodscomprise administering to the mammal an amount of a grape producteffective to reduce or prevent oxidative stress in the mammal's liver.In some embodiments, the method reduces or prevents lipid peroxidationin the liver.

In some embodiments, methods of protecting, supporting, enhancing, ormaintaining cytochrome P450 and/or Phase II enzymatic detoxificationsystem in a mammal's liver are provided, wherein the methods compriseadministering to the mammal an amount of a grape product effective toprotect, support, enhance, or maintain cytochrome P450 and/or Phase IIenzymatic detoxification system in the mammal's liver.

In some embodiments, methods of upregulating expression of genes thatcontrol production of detoxification enzymes in a mammal are provided,wherein the methods comprise administering to the mammal an amount of agrape product effective to upregulate expression of genes that controlproduction of detoxification enzymes in the mammal. In some embodiments,the gene is NRF2.

In some embodiments, methods of increasing production of antioxidants ina mammal are provided, wherein the methods comprise administering to themammal an amount of a grape product effective to increase the productionof antioxidants in the mammal. In some embodiments, the antioxidantincreased is glutathione, superoxide dismutase, or catalase. In thevarious embodiments described herein, the mammal or subject can be amammal or a subject in need thereof. In some embodiments, the mammal hasbeen identified as having non-alcoholic fatty liver disease or relatedconditions, which are described herein. In some embodiments, the grapeproduct reduces the amount of fat deposited in the liver in the mammal.

As described herein, the grape seed product can be a grape seed flour.Accordingly, in some embodiments, the seed flour can also be aChardonnay seed flour. In some embodiments, the grape seed product is agrape seed extract. In some embodiments, the extract is a Chardonnayseed extract.

As described herein, the composition can contain a grape product and atleast one additional compound. In some embodiments, the compound is anantioxidant. In some embodiments, the at least one additional compoundis vitamin E, cinnamon, black bear bile, obeticholic acid,2,4-dinitrophenol (DNP), DNP-methyl ether (DNPME), an anti-glycemiccompound, or an anti-cholesterol compound. In some embodiments, the atleast one additional compound is vitamin A, vitamin C, a carotenoid,lipoic acid, ubiquinol, ubiquinone, or glutathione. In some embodiments,the at least one additional compound is a second type of grape seedproduct. That is, the composition can comprise grape seed compositionsderived from two or more different types of grapes. The types of grapescan be any type, such as those, but not limited to, the grapes describedherein.

In some embodiments, the anti-glycemic compound is an insulinsensitizer. Examples of insulin sensitizers include, but are not limitedto, pioglitazone, metformin, sulfonylureas, or thiazolidinediones.

In some embodiments, the anti-cholesterol compound is a HMG-CoAreductase inhibitor. These can sometimes be referred to colloquially as“statins.” In some embodiments, the HMG-CoA reductase inhibitor isatorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin,rosuvastatin, or simvastatin.

In some embodiments, the method comprises administering the grapeproduct and the additional product concurrently or in series. Theproducts can, therefore, be administered sequentially or simultaneously.In some embodiments, the grape product and the at least one additionalcompound or composition are combined into the same formulation or unitdosage form and administered together. The unit dosage form, asdescribed herein, can be, for example, a powder, capsule or tablet. Insome embodiments, the different compositions are administered inseparate formulations.

Formulations comprising a grape product and at least one additionalcompound or composition are also provided. In some embodiments, the atleast one additional compound or composition is a second type of grapeproduct. In some embodiments, the composition comprises a grape productand at least one additional compound. In some embodiments, it is anantioxidant. In some embodiments, the at least one additional compoundis vitamin E, obeticholic acid, 2,4-dinitrophenol (DNP), DNP-methylether (DNPME), an anti-glycemic compound, or an anti-cholesterolcompound. In some embodiments, the at least one additional compound isvitamin A, vitamin C, a carotenoid, lipoic acid, ubiquinol, ubiquinone,or glutathione. In some embodiments, the at least one additionalcompound is a methyl donor, such as, but not limited to,s-adenosyl-methionine, trimethyl glycine, or methionine. In someembodiments, the at least one additional compound is a metabolicnutrient, such as, but not limited to, a phosphatide, an essential fattyacid, choline, or a B vitamin. In some embodiments, the at least oneadditional compound is a botanical or a botanical extract, such as, butnot limited to, milk thistle, schizandra, burdock dandelion, artichoke,turmeric, celandine, kudzu, chicory, or yellow dock, or any extractthereof. Examples of the anti-glycemic compound or an anti-cholesterolcompound are described herein. For example, in some embodiments, theanti-glycemic compound is an insulin sensitizer. In some embodiments,the insulin sensitizer is pioglitazone, metformin, thiazolidinediones,or sulfonylureas. In some embodiments, the composition comprises ananti-cholesterol compound is a HMG-CoA reductase inhibitor. In someembodiments, the HMG-CoA reductase inhibitor is atorvastatin,fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, orsimvastatin.

In some embodiments, the composition is suitable for mammal (e.g. human,livestock, husbanded animal, pet) consumption. In some embodiments, thecomposition is suitable for cattle, poultry, dogs, cats, and the like.In some embodiments, the compositions described herein are combined witha probiotic. Therefore, in some embodiments, in the methods describedherein, the subject is a mammal (e.g. human, livestock, husbandedanimal, pet). In some embodiments, the subject is cattle, poultry, dogs,cats, and the like.

In some embodiments, the compositions described herein can be used toincrease insulin resistance. In some embodiments, the compositionsdescribed herein can be used to maintain normal (e.g. healthy) insulinresistance. In some embodiments, the compositions described herein canbe used to maintain a healthy level of hepatic leptin. In someembodiments, the compositions described herein can be used to restorehepatic leptin to healthy or normal levels. In some embodiments, thecompositions described herein can be used to improve, maintain, orrestore hepatic leptin sensitivity in a mammal, including a mammal inneed thereof. Accordingly, the compositions can be used to reducehepatic steatosis.

In some embodiments, the compositions can be used to reduce the liverand/or epididymal adipose tissue weights by 30-40% as compared to amammal on a diet without the grape product (e.g. seed extract or flour).In some embodiments, the compositions are used to reduce the hepaticlipid content by about 40-50% in a mammal on a high fat diet as comparedto a mammal on a control diet. In some embodiments, the compositions areused to reduce the hepatic lipid content in a mammal. The compositionscan be administered according to the various methods described hereinin, for example, an amount described herein.

Embodiments disclosed herein also provide methods of modulating theexpression of one or more of the genes described herein. In someembodiments, the methods comprise administering a grape extract (e.g.seed extract, pomace, or flour) to a mammal expressing one or more ofthe genes. In some embodiments, methods of modulating the expression ofa gene in a mammal comprising administering a grape seed extract to themammal in an effective amount to modulate the expression of the gene areprovided. In some embodiments the gene expression is increased. In someembodiments, the gene expression is decreased. In some embodiments, themammal is a mammal in need of modulated gene expression. In someembodiments, the expression of the gene is increased or decreased atleast 1.5 fold as compared to the mammal not administered the grape seedextract. In some embodiments, the expression of the gene is increaseddecreased at least 2, 3, 4, 5, or 6 fold. In some embodiments, theexpression of the gene is increased or decreased about 1.5 to 6, 1.5 to5, 1.5 to 4, 1.5 to 3, 1.5 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6,3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6, or 6 to 7 fold. In someembodiments, the gene expression is modulated according to the amountsshown in the Examples.

In some embodiments, the gene is one or more of Chi3l1, Ces1d, Adra1b,Slc13a3, Ntrk2, Cyp21a1, Aqp8, Gck, Pfkm, Txn1, Ckb, Gckr, Fen1,Cyp46a1, Vldlr, Ppcdc, Bdh2, Scd1, Acot11, Mlxip1 (ChREBP), Lcn13,Prodh, Gstt3, Mc5r, Tas2r104, Vmn1r192, Ras12-9, Cfd, Ctse, Orm2, Rorc,Tlr5, Hnmt, Cyp2b13, Cyp2d40, Hao2, Gdf15, Sptlc3, Mogat1, Plin4, Ifna9,Asns, Got1, Atp6v0d2, Cyp7b1, Cyp17a1, Id1, Avpr1a, Ocln, Cyp51, Fdft1,Hmgcr, Hsd17b7, Insig1, Lss, Mvd, Mvk, Nsdhl, Sc4mol, Tm7sf2, Apom,Lepr, Pcsk9, Sqle, Il17rb, Acsl3, Agxt, Aldoc, Pgk2, Tlr13, Hmgcs1,Stard4, Mmp7, and Igfbp2. In some embodiments, the gene is 2, 3, 4, or 5of the preceding list. In some embodiments, the gene is each of thegenes of the preceding list.

Embodiments provided herein also include, but are not limited to:

-   -   1. A method of treating or preventing non-alcoholic fatty liver        disease (NAFLD) in a mammal comprising administering to the        mammal an amount of a grape product effective to treat or        prevent NAFLD.    -   2. A method of reducing hepatic steatosis in a mammal comprising        administering to the mammal an amount of a grape product        effective to reduce hepatic steatosis.    -   3. A method of reducing steatohepatitis in a mammal comprising        administering to the mammal an amount of a grape product        effective to reduce steatohepatitis.    -   4. A method of reducing hepatic fibrosis in a mammal comprising        administering to the mammal an amount of a grape product        effective to reduce hepatic fibrosis.    -   5. A method of treating or preventing non-alcoholic        steatohepatitis (NASH) in a mammal comprising administering to        the mammal an amount of a grape product effective to treat or        prevent NASH.    -   6. A method of supporting a healthy liver in a mammal comprising        administering to the mammal an amount of a grape product        effective to support a healthy liver.    -   7. The method of embodiment 6, wherein the mammal has been        identified as having non-alcoholic fatty liver disease.    -   8. A method of reducing or preventing oxidative stress in a        mammal's liver comprising administering to the mammal an amount        of a grape product effective to reduce or prevent oxidative        stress in the mammal's liver.    -   9. The method of embodiment 8, wherein the method reduces or        prevents lipid peroxidation.    -   10. A method of protecting, supporting, enhancing, or        maintaining cytochrome P450 and/or Phase II enzymatic        detoxification system in a mammal's liver comprising        administering to the mammal an amount of a grape product        effective to protect, support, enhance, or maintain cytochrome        P450 and/or Phase II enzymatic detoxification system in the        mammal's liver.    -   11. A method of upregulating expression of genes that control        production of detoxification enzymes in a mammal, the method        comprising administering to the mammal an amount of a grape        product effective to upregulate expression of genes that control        production of detoxification enzymes in the mammal.    -   12. The method of embodiment 11, wherein the gene is NRF2.    -   13. A method of increasing production of antioxidants in a        mammal, the method comprising administering to the mammal an        amount of a grape product effective to increase the production        of antioxidants in the mammal.    -   14. The method of embodiment 13, wherein the antioxidant is        glutathione, superoxide dismutase, or catalase.    -   15. The method of any one of embodiments 1-14, wherein the grape        product reduces the amount of fat deposited in the liver.    -   16. The method of any one of embodiments 1-15, wherein the        mammal is a mammal in need thereof    -   17. The method of any one embodiments 1-16, wherein the grape        product is whole pomace or a portion thereof, a grape seed        product, a grape skin product, or any extract thereof, or any        combination thereof    -   18. The method of embodiment 17, wherein the grape seed product        is grape seed flour.    -   19. The method of embodiment 17, wherein the grape product is a        grape skin extract, grape skin flour, or a grape skin powder.    -   20. The method of embodiment 17, wherein the grape product is a        pomace extract.    -   21. The method of embodiment 17, wherein the grape product is        grape seed extract.    -   22. The method of embodiment 21, wherein the grape extract is a        methanol or ethanol extract.    -   23. The method of any one of embodiments 1-22, wherein the grape        product is administered in effective amount of about 1 mg to        about 15,000 mg.    -   24. The method of any one of embodiments 1-23, wherein the        mammal is a human, companion animal, domestic pet, husbanded        animal, or livestock.    -   25. The method of any one of embodiments 1-24, wherein the        method further comprises at least one additional compound.    -   26. The method of embodiment 25, wherein the at least one        additional compound is an antioxidant, an anti-cholesterol        medication, anti-glycemic compound, or an insulin sensitizer.    -   27. The method of embodiment 25, wherein the at least one        additional compound is vitamin E, vitamin A, vitamin C, a        carotenoid, lipoic acid, ubiquinol, ubiquinone, or glutathione.    -   28. The pharmaceutical composition of embodiment 25, wherein the        at least one additional compound is s-adenosyl-methionine,        trimethyl glycine, or methionine.    -   29. The pharmaceutical composition of embodiment 25, wherein the        at least one additional compound is a phosphatide, an essential        fatty acid, choline, or a B vitamin.    -   30. The pharmaceutical composition of embodiment 25, wherein the        at least one additional compound is a botanical or an extract        thereof    -   31. The pharmaceutical composition of embodiment 30, wherein the        botanical or an extract thereof, is milk thistle, schizandra,        burdock, dandelion, artichoke, turmeric, celandine, kudzu,        chicory, or yellow dock, or any extract thereof    -   32. The method of embodiment 26, wherein the anti-glycemic        compound is an insulin sensitizer.    -   33. The method of embodiment 32, wherein the insulin sensitizer        is pioglitazone, metformin, sulfonylureas, or        thiazolidinediones.    -   34. The method of embodiment 26, wherein the anti-cholesterol        compound is a HMG-CoA reductase inhibitor.    -   35. The method of embodiment 34, wherein the HMG-CoA reductase        inhibitor is atorvastatin, fluvastatin, lovastatin,        pitavastatin, pravastatin, rosuvastatin, or simvastatin.    -   36. The method of embodiment 25, wherein the grape product and        the additional compound are administered in concurrently or in        series.    -   37. The method of embodiment 25, wherein the grape product and        the additional compound are administered in the same formulation        or in different formulations.    -   38. The method of any one of embodiments 1-37, wherein the        mammal is on a high fat diet.    -   39. The method of any one of embodiments 1-37, where the mammal        is on a diet comprising 30 to 50% fat by calorie content.    -   40. The method of any one of embodiments 1-39, wherein the grape        is a Chardonnay grape.    -   41. The method of any one of embodiments 1-39, wherein the grape        is a Cabernet Sauvignon grape, a Pinot Noir grape, a Sauvignon        Blanc grape, or a White Riesling grape.    -   42. A pharmaceutical composition comprising a grape product and        at least one additional compound.    -   43. The pharmaceutical composition of embodiment 42, wherein the        grape product is whole pomace or a portion thereof, a grape seed        product, a grape skin product, or any combination thereof, or        any extract thereof.    -   44. The pharmaceutical composition of embodiment 43, wherein the        grape seed product is a grape seed flour.    -   45. The pharmaceutical composition of embodiment 43, wherein the        grape skin product is a grape skin extract.    -   46. The pharmaceutical composition of any one of embodiments        42-45, wherein the grape is a Chardonnay grape, Cabernet        Sauvignon grape, Pinot Noir grape, Sauvignon Blanc grape, or a        White Riesling grape product.    -   47. The pharmaceutical composition of embodiment 42, wherein the        at least one additional compound is an antioxidant.    -   48. The pharmaceutical composition of embodiment 42, wherein the        at least one additional compound is vitamin E, obeticholic acid,        2,4-dinitrophenol (DNP), DNP-methyl ether (DNPME), an        anti-glycemic compound, or an anti-cholesterol compound.    -   49. The pharmaceutical composition of embodiment 42, wherein the        at least one additional compound is vitamin E, vitamin A,        vitamin C, a carotenoid, lipoic acid, ubiquinol, ubiquinone, or        glutathione.    -   50. The pharmaceutical composition of embodiment 42, wherein the        at least one additional compound is s-adenosyl-methionine,        trimethyl glycine, or methionine.    -   51. The pharmaceutical composition of embodiment 42, wherein the        at least one additional compound is a phosphatide, an essential        fatty acid, choline, or a B vitamin.    -   52. The pharmaceutical composition of embodiment 42, wherein the        at least one additional compound is a botanical or an extract        thereof.    -   53. The pharmaceutical composition of embodiment 52, wherein the        botanical or an extract thereof, is milk thistle, schizandra,        burdock, dandelion, artichoke, turmeric, celandine, kudzu,        chicory, or yellow dock, or any extract thereof    -   54. The pharmaceutical composition of embodiment 48, wherein the        anti-glycemic compound is an insulin sensitizer.    -   55. The pharmaceutical composition of embodiment 54, wherein the        insulin sensitizer is pioglitazone, metformin,        thiazolidinediones, or sulfonylureas.    -   56. The pharmaceutical composition of embodiment 48, wherein the        anti-cholesterol compound is a HMG-CoA reductase inhibitor.    -   57. The pharmaceutical composition of embodiment 56, wherein the        HMG-CoA reductase inhibitor is atorvastatin, fluvastatin,        lovastatin, pitavastatin, pravastatin, rosuvastatin, or        simvastatin.    -   58. A method of modulating the expression of one or more of the        genes in Table 3 comprising administering a grape product to a        mammal expressing one or more of the genes.    -   59. The method of embodiment 58, wherein the gene is one or more        of Chi3l1, Ces1d, Adra1b, Slc13a3, Ntrk2, Cyp21a1, Aqp8, Gck,        Pfkm, Txn1, Ckb, Gckr, Fen1, Cyp46a1, Vldlr, Ppcdc, Bdh2, Scd1,        Acot11, Mlxip1 (ChREBP), Lcn13, Prodh, Gstt3, Mc5r, Tas2r104,        Vmn1r192, Rasl2-9, Cfd, Ctse, Orm2, Rorc, Tlr5, Hnmt, Cyp2b13,        Cyp2d40, Hao2, Gdf15, Sptlc3, Mogat1, Plin4, Ifna9, Asns, Got1,        Atp6v0d2, Cyp7b1, Cyp17a1, Id1, Avpr1a, Ocln, Cyp51, Fdft1,        Hmgcr, Hsd17b7, Insig1, Lss, Mvd, Mvk, Nsdhl, Sc4 mol, Tm7sf2,        Apom, Lepr, Pcsk9, Sqle, Il17rb, Acsl3, Agxt, Aldoc, Pgk2,        Tlr13, Hmgcs1, Stard4, Mmp7, and Igfbp2.    -   60. The method of embodiment 58, wherein the grape product is a        grape seed flour extract.    -   61. The method of embodiment 58, wherein the grape product is a        grape seed flour.    -   62. The method of any one of embodiments 58-61, wherein the        grape is a Chardonnay grape.    -   63. The method of any one of embodiments 58-61, wherein the        grape is a Cabernet Sauvignon, Pinot Noir grape, Sauvignon Blanc        grape, or a White Riesling grape.

The embodiments will not be further described with reference to theexamples, which are non-limiting.

EXAMPLES Example 1

Diet-induced (DIO) hamsters were fed a high fat (HF) diet for 10 weeks.Animals that exhibited low glucose tolerance and were in the heaviestcategory were given the various treatments. Animals were fed dietscontaining partially defatted Chardonnay flavonoid-rich wine grape seedflour (ChrSd), commercial extracts, or laboratory produced ethanolicextracts of ChrSd for 10 and 20 weeks, respectively. Half weresacrificed and examined for signs of hepatic dysfunction at 10 weeks andthe other half examined after 20 weeks. Animals on treatments benefited.Animals on ChrSd whole flour treatments showed no signs of ectopic fatin the liver, whereas those on the HF Control diet showed extensive fatdeposition in the liver. Livers of animals under various treatmentsweighed significantly less than those on the HF Control. Blood lipidprofiles of animals on various grape seed treatments showed improvedblood lipid profiles. Accordingly, ChrSd can be used to maintain ahealthy blood lipid profile, such as when the animal is subjected to ahigh fat diet.

Example 2

Diet-induced obese (DIO) mice were fed high-fat (HF) diets containingeither partially defatted flavonoid-rich Chardonnay grape seed flour(ChrSd) or microcrystalline cellulose (MCC, control) for 5 weeks inorder to determine whether ChrSd improved insulin resistance and thepathogenesis of hepatic steatosis. The 2-h insulin and glucose areasunder the curves were significantly lowered by ChrSd, indicating thatChrSd improved insulin sensitivity and glucose metabolism. ChrSd intakealso significantly reduced body weight gain, liver and adipose tissueweights, hepatic lipid content, and plasma low-density lipoprotein(LDL)-cholesterol, despite a significant increase in food intake. Exonmicroarray analysis of hepatic gene expression revealed down-regulationof genes related to triglyceride and ceramide synthesis, immuneresponse, oxidative stress, and inflammation, and up-regulation of genesrelated to fatty acid oxidation, cholesterol, and bile acid synthesis.Expression of leptin receptor was up-regulated, suggesting enhancedhepatic leptin sensitivity. Pathway analysis of the microarray datarevealed that lipid and cholesterol metabolism, and infectious andmetabolic disease pathways were differentially regulated by ChrSd. Inconclusion, ChrSd ameliorated the effects of a HF diet on weight gain,insulin resistance, and progression of hepatic steatosis in DIO mice viamodulation of hepatic expression of genes related to oxidative stress,inflammation, and lipid and cholesterol metabolism.

Animals and Diets. Male C57BL/6J mice were housed individually in anenvironmentally controlled room (20-22C, 60% relative humidity, 12-halternating light/dark cycle). The mice were acclimated and given adlibitum access to water and mouse chow diet (LabDiet 5015, PMIInternational, Redwood, Calif., USA) for 1 week prior to initiation ofthe experimental diets. Mice were weighed and randomized into two groupsof 30 mice each. Mice were fed ad libitum with either mouse chow diet ora HF diet containing 17% of energy as protein, 37% as carbohydrate, and47% as fat, with 0.1% cholesterol. After 5 weeks, mice were weighed, anddiet-induced obese (DIO) mice were identified as those having gainedsignificantly more weight than the chow-fed mice. The DIO mice were thenrandomized into two groups (n=10 each) and fed ad libitum for 5 weekswith HF diets containing either 10% ChrSd (Sonomaceuticals,LLC/WholeVine Products, Santa Rosa, Calif.) or 5% microcrystallinecellulose (MCC, control diet; Dyets Inc., Bethlehem, Pa.) (Table 1).MCC, an insoluble fiber, has little effect on sterol metabolism (Horton,1994 #56). Chardonnay grape pomace was obtained from coastal vineyardsin Sonoma County, California. Seeds from the 2010 vintage were driedusing heated air (55-70C) and separated from skins and stems. Theresidual press cake was milled to pass through an 85 mesh sieve, afteroil had been pressed from the seed. The total flavonoids, totalcatechins, catechin, and epicatechin contents of ChrSd were 12,000,1610, 701, and 732 mg/100 g, respectively. Body weights were recordedweekly, and food intake was monitored twice per week. The studyprotocol, #P-04-02, was approved by the Animal Care and Use Committee,Western Regional Research Center, USDA, Albany, Calif., USA.

Plasma and Liver Collection. Mice were feed-deprived for 12 h andanesthetized with isoflurane (Phoenix Pharmaceutical, St. Joseph, Mo.,USA). Blood was collected by cardiac puncture with syringes previouslyrinsed with potassium EDTA solution (15% w/v). The plasma was separatedafter centrifugation at 2,000×g for 30 min at 4 C. Livers and epididymaladipose tissues were collected, weighed, and immediately frozen inliquid nitrogen for later analysis. After freeze-drying, the powderedlivers were weighed and mixed with 2 mL of CHCl₃/MeOH (2:1), sonicatedfor 5 min, and then incubated overnight. The samples were centrifugedfor 10 min at 1000 rpm, and the supernatant was removed. Another 2 mL ofCHCl₃/MeOH was added, sonicated, and allowed to stand overnight toextract. Solvent was removed from the combined extracts under nitrogenand the total hepatic total lipid content was determinedgravimetrically.

Plasma Lipid Analysis. Plasma lipoprotein cholesterol was determined bysize exclusion chromatography. Briefly, high-performance liquidchromatograpy (HPLC) was carried out using an Agilent 1100 HPLCchromatograph with a Superose 6HR HPLC column (Pharmacia LKBBiotechnology, Piscataway, N.J., USA) consisting of a mixing coil(1615-50 Bodman, Aston, Pa., USA) in a temperature-controlled waterjacket (Aura Industrials, Staten, N.Y., USA). A Hewlett-Packard HPLCpump (79851-A; Agilent Technologies, Palo Alto, Calif., USA) was used todeliver cholesterol reagent (Roche Diagnostics, Indianapolis, Ind., USA)at a flow rate of 0.2 mL/min. Bovine cholesterol lipoprotein standardswere used to calibrate the signal on the basis of peak areas.

Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT). After a3-h fast, mice were administrated glucose intraperitoneally (2 g/kg bodyweight), and tail vein blood glucose levels were determined at 0, 15,30, 60, and 120 min after glucose injection using a OneTouch Ultrameter(LifeScan Inc., Wayne, Pa.). ITT was performed after mice wereadministrated insulin intraperitoneally (0.5 U/kg body weight). Glucoselevels were determined in tail vein blood at 0, 30, and 60 min afterinsulin injection using a OneTouch Ultrameter (LifeScan Inc.).

Gene Expression and Exon Microarray Analysis. Total liver RNA wasextracted from three biological replicates within each group using aTRIzolplus RNA purification kit (Invitrogen, Life Technologies,Carlsbad, Calif., USA). The total RNA quality was determined using a2100 Bioanalyzer instrument and RNA 6000 Nano LabChip assay (AgilentTechnologies, Palo Alto, Calif., USA). Total RNA (10 μg) was then usedto synthesize one-cycle cDNA (first-strand and second-strand cDNAsynthesis) followed by clean-up of double-stranded cDNA, andbiotin-labeled cRNA synthesis. The biotin-labeled cRNA was fragmentedusing One-Cycle Target Labeling and Control reagents (Affymetrix, SantaClara, Calif., USA). Fragmented cRNA samples were hybridized to anAffymetrix GeneChip Mouse exon 1.0 ST array, an expression and exonsplicing array containing 1.2 million probe sets, representing 80000genes. The hybridization signals were acquired and analyzed using theGeneChip Scanner 3000 High-Resolution Scanner (Affymetrix) and theAffymetrix GeneChip Operating Software (GCOS). Analysis of both geneexpression and exon alternative splicing from the microarray data wasperformed using a GeneSpring GX version 11.0 program (AgilentTechnologies, Santa Clara, Calif.). Gene expression was determined to besignificant when the change was found to be 1.5-fold and above. Thesplice index was defined as the log of the ratio of exon-levelexpression over gene-level expression. A fold change in splice indexvalue ≥2 between treatment and control groups was considered to bedifferentially spliced. Transcripts with at least one differentiallyspliced exon were considered to be differentially regulated splicing.

Statistical Analysis. All data were expressed as means±SE. Analysis ofvariance (ANOVA) was performed using the JMP7 statistical program (SASInstitute, Cary, N.C., USA) to examine the effects of treatment onplasma lipid levels, body and tissue weights, total energy intake, andfeed efficiency ratio. Significance was defined at P<0.05. IngenuityPathways Analysis tool (IPA version 8.7, Ingenuity Systems Inc., RedwoodCity, Calif., USA; http://www.ingenuity.com) was used to analyze theexon microarray data determining biological mechanisms, pathways, andfunctions from the differentially expressed genes. Right-tailed Fisher'sexact test was used to calculate the P value. P values represent theprobability that the biological function of each data set, biologicalfunction and disease assigned to a particular network for each data set,and the association between the genes in a data set and thecorresponding canonical pathway were explained by chance.

Metabolic Effects.

ChrSd supplementation of a HF diet for 5 weeks significantly loweredbody weight gain of the DIO mice despite a significant increase of totalenergy intake, resulting in a 72% lower energy efficiency ratio in thesemice (Table 2). The ChrSd supplemented diet significantly lowered theliver and epididymal adipose tissue weights by 38% and 35%,respectively, as compared with mice on the control diet (Table 2). Totalhepatic lipid content was 43% lower in DIO mice fed ChrSd, as comparedto control diet (P<0.05) (Table 2). Dietary ChrSd supplementationsignificantly lowered peak blood glucose response at 60 min (P<0.05) andthe area under the curve (AUC) during a 2-h glucose response (FIGS. 2Aand B). ChrSd supplementation also resulted in a marked reduction ofinsulin response at 30 min (P<0.05) and 60 min (P<0.05) (FIGS. 3A andB).

Microarray Analysis of Hepatic Gene Expression Profiles

The comprehensive expression of hepatic genes in DIO mice fed HF dietssupplemented with either 5% MCC or 10% ChrSd flour was assessed by exonmicroarray analysis. A number of genes were differentially expressed inmice fed 10% ChrSd, as compared to those fed 5% MCC (P<0.05, fold change≥1.5) (Table 3). Among these genes, some were down-regulated and somewere up-regulated. Table 3 shows the genes differentially down- andup-regulated by ChrSd, categorized by biological process. Chitinase-like1(Chi3l1; fold change, −1.5), encoding a protein involved in theactivation of nuclear factor-kappaB (NF-κB)-induced kinase activity,involved in inflammation and tissue remodeling, was down-regulated. Agene encoding an enzyme involved in cortiscosteroid biosynthesis(C21-steroid hormone biosynthesis, cytochrome P450, family 21, subfamilya polypeptide 1 (Cyp21a1; fold change, −1.6)) was down-regulated. Theexpression level of aquaporin 8 (Aqp8; fold change, −1.8), encoding aprotein related to canalicular bile acid transport was down-regulated.Genes encoding proteins involved in diacylglycerol and triacylglycerolbiosynthetic processes (stearoyl-coenzyme A desaturase 1 [Scd1];monoacylglycerol O-acyltransferase 1 [Mogat1]) were down-regulated (foldchange, −1.6 for both). Expression of odorant binding protein 2A(Lcn13), involved in glucose and lipid metabolism, was down-regulated(fold change, −3.5). Genes involved in immune system processes,including complement factor D (Cfd; fold change, −2.4), cathepsin E(Ctse; fold change, −1.7), orosomucoid 2 (Orm2, fold change, −1.7),retinoic acid receptor-related orphan receptor gamma (Rorc; fold change−1.5), and toll-like receptor 5 (Tlr5; fold change, −2) weredown-regulated. Serine palmitoyltransferase, long chain base subunit 3(Sptlc3; fold change, −1.7), which is involved in sphingolipidmetabolism, was down-regulated. Expression of a lipid droplet-associatedprotein involved in triglyceride metabolic process, perilipin 4 (Plin4;fold change, −3.5), was also down-regulated in mice fed ChrSd, ascompared to mice fed MCC. Interferon alpha 9 (Ifna9; fold change, 1.8),a gene encoding a protein related to host immune defense response, wasup-regulated. The expression of genes encoding cytochrome P450, family7, subfamily b, polypeptide 1 (Cyp7b1; fold change, 1.6) and cytochromeP450, family 17, subfamily a, polypeptide 1 (Cyp17a1; fold change, 2.5),related to bile acid metabolism, was up-regulated. Genes involved incholesterol metabolism, including sterol 14-demethylase (Cyp51; foldchange, 5.6), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr;fold change, 2.5), hydroxysteroid (17-β) dehydrogenase 7 (Hsd17b7; foldchange, 2.8), insulin induced gene 1 (Insig1; fold change, 1.8),sterol-C4-methyl oxidase-like (Sc4mol; fold change, 4.5), and leptinreceptor (Lepr; fold change, 1.6) were up-regulated. The expressionlevel of acyl-CoA synthetase long-chain family member 3 (Acsl3), whichis involved in fatty acid β-oxidation, was up-regulated.

Pathway analysis using the IPA System identified several biologicalfunctions and canonical gene pathways that were differentially regulatedby ChrSd supplementation (Table 4). When grouped by biological function,the expression levels of genes related to lipid metabolism,hematological disease, and metabolic disease were significantly affectedby ChrSd supplementation. In terms of canonical pathways, ChrSd affectedthe cholesterol biosynthesis superpathway and the zymosterolbiosynthetic pathway. Networks involving lipid metabolism and infectiousdisease were affected by ChrSd supplementation. Insig1, which regulatescholesterol concentration, was identified as a major regulator effectsnetworks (data not shown). Analysis of exon microarray data using theGeneSpring GX11.0 program resulted in the identification of 84 geneswith SI>|2.0| in the presence of ChrSd supplementation (data not shown).Further analysis by using RT-PCR needs to be done to confirm thealternative splicing of these genes.

In the current study, exon microarray analysis was used to identifydifferentially expressed hepatic genes in DIO mice, to provide novelbiological insights into the effects of ChrSd supplementation on HFdiet-induced hepatic steatosis. We found that, in addition to affectingexpression of genes involved in cholesterol, bile acid, and lipidmetabolism, dietary ChrSd supplementation significantly affected thehepatic expression of genes involved in C21-steroid metabolism, immunesystem processes, inflammation, tissue remodeling, and lipid storage.ChrSd supplementation markedly up-regulated hepatic expression of genesrelated to bile acid and cholesterol synthesis (Cyp17a1, Cyp51, Hmgcr,and Insig1), and fatty acid β-oxidation (Acsl3), whereas itsignificantly down-regulated the expression of genes related to fattyacid biosynthesis (Scd1, Acot11, and Mlxip1/carbohydrate responsiveelement-binding protein [ChREBP]), triacylglycerol biosynthesis(Mogat1), oxidative stress (Growth differentiation factor 15, Gdf15),inflammatory and immune processes (Cfd, Chi3l1, Ctse, Orm2, Rorc, andTlr5), ceramide biosynthesis (Sptlc3), and lipid storage (Plin4). Thesealtered gene expression profiles in hepatic tissue were accompanied bysignificant reductions in plasma LDL-cholesterol concentration, liver,adipose, and body weights, feed efficiency ratio, and insulinresistance, as compared to the MCC supplemented control group. Notably,ChrSd supplementation lowered the hepatic expression of genes that havea role in obesity including Insig-1, lepR, Neurotrophic tyrosine kinase,receptor, type 2 (Ntrk2), Melanocortin 5 receptor (Mc5r), and Matrixmetallopeptidase 7 (Mmp7).

Without being bound by any particular theory, we hypothesize thatsupplementation with ChrSd containing high amounts of flavonoids canup-regulate genes related to scavenging of reactive oxygen species (ROS)and free radicals, and down-regulate genes related to oxidative stress,inflammation, and fatty acid biosynthesis, leading to improvedHF-induced insulin resistance and NAFLD. The suggested mechanismsinvolved in the antioxidant activity of flavonoids are: 1) free radicalscavenging and metal chelating activities; 2) cell-to-cell signalingpathways; and 3) antioxidant enzyme gene expression. The antioxidantactivity of grape seed products has been monitored in in vitro. The invivo biological significance of in vitro activity is not clear becauseoligomers and larger flavonoids are poorly absorbed, while monomers arerapidly metabolized and cleared from the body. In the present study,expression of stress responsive genes (Gdf15 and ChREBP) wasdown-regulated following ChrSd supplementation. The hepatic expressionof Gdf15, a member of the transforming growth factor (TGF)β superfamily,is related to hepatic steatosis under ER stress. Oxidative stressactivates ChREBP, which transcriptionally modulates lipogenic andglycolytic genes, resulting in fatty liver. Taken together, thesefindings indicated that the flavonoid-rich ChrSd supplement canpotentiate antioxidant activity and reduce oxidative stress, resultingin transcriptional down-regulation of ChREBP and lipogenic genes (Scd1,Acot11, Mogat1) in the liver. Despite the down-regulation of stressrelated genes, the canonical pathway analysis did not identifysignificant changes in the free radical and ER stress-related signalingpathways in the livers of DIO mice supplemented with ChrSd. Oxidativestress and inflammation are closely interlinked in obesity. However,whether improved oxidative stress and inflammation are secondary to orindependent of reduction in hepatic lipid levels is not clear.Additionally, we cannot exclude the possibility that C57BL/6J mice fed aHF diet (47% of energy as fat) for 10 weeks may have already passedthrough the obesity-related ER stress response and entered thelipogenesis stage. This transition from ER stress to lipogenesis wasshown in a previous study of ApoE3L mice on HF diets for 16 weeks; theseanimals exhibited an early phase hepatic response to the HF diet thatwas characterized by changes in genes linked to the stress pathways,followed by a late phase involving genes related to lipid accumulation.Likewise, the present study found few ChrSd-induced changes of genesrelated to free radicals and ER-stress response.

The C57BL/6J mouse has been used extensively to study hepatic steatosis.This animal model shows more susceptibility to HF diet-induced fattyliver disease than BALB/c mice. Genes related to the 26S proteasome andubiquitin proteasome system in wild type C57BL/6J mice fed HF diets (40%energy as fat) for 15 weeks were up-regulated relative to transgenicmice with lowered proteasome activity, resulting in obesity and hepaticsteatosis. In contrast, no significant modulation of genes related toER-stress and inflammation was observed. In another study, C57BL/6J micefed a HF diet (60% of energy as fat) developed hepatic steatosis andinflammation after 24 weeks. In the present study, we selected obesityresponsive mice by feeding HF diet for 5 weeks and then selecting DIOmice for treatment with HF diets supplemented with either ChrSd or MCC(control) for an additional 5 weeks. The lower degree of free radicalscavenging response may be due to the shorter duration of the ChrSdsupplementation phase of the study and the lower level of dietary fat,as compared to previous studies showing an oxidative stress response.

Flavonoids have been reported to show immunomodulatory andanti-inflammatory properties, although most of these studies have beenconducted in vitro, with very few in vivo or human analyses. In humans,the consumption of 600 mg/day grape seed extract for 4 weeks improvedmarkers of insulin resistance (e.g. blood C-reactive protein [CRP]concentration) and inflammation in type 2 diabetic patients at high riskfor cardiovascular disease. In this study, ChrSd supplementationdown-regulated hepatic expression of genes related to immune andinflammation processes (Cfd, Chi3l1, Ctse, Orm2, Rorc, Tlr5). Cfd(adipsin) is a serine protease involved as a host response factor in theremoval of foreign antigens and pathogens. The Chi3l1 gene is related toactivation of NF-κB-induced kinase activity and inflammation. Ctse is anintracellular aspartic proteinase highly expressed in immune-relatedcells, such as macrophages, and its main role is related to macrophageinfiltration, adipogenesis, and hepatic steatosis. Orm2 is expressed inhepatocytes and adipocytes, and secreted into plasma under metabolic andinflammatory stress as an acute phase reactant immunomodulator protein.Rorc regulates Th17 cell differentiation, controlling the production ofinflammatory cytokines. Tlr5 is predominantly expressed in epithelialcells of the intestinal mucosa and its expression in liver and adiposetissue is relatively very low. Interestingly, HF diet has been shown toincrease Tlr5 expression in mouse epididymal adipose tissue. It isactivated by bacterial flagellin proteins and triggers innate immuneresponses and NF-κB. Although the biological significance of hepaticTlr5 down-regulation is unclear, ChrSd supplementation may reduce thegut bacterial residence-derived inflammatory response to HF-inducedstress. Our previous study suggested that hepatic cholesterol metabolismwas influenced by the composition of intestinal microbiota and byintestinal fibroblast growth factor (FGF) 15 gene expression in hamstersfollowing ChrSd supplementation. Collectively, these findings indicatethat ChrSd supplementation may prevent HF diet-induced inflammation inthe liver via down-regulation of hepatic expression of genes related toimmune/inflammatory pathways.

ChrSd supplementation up-regulated hepatic leptin receptor expression by1.5-fold, as compared with control. Leptin, a hormone produced byadipose tissue, regulates energy intake and expenditure and enhancesfatty acid oxidation in liver and muscle. In DIO mice, down-regulationof hepatic leptin receptor expression was observed, suggesting thatobesity induces hepatic insensitivity to leptin. Thus, theChrSd-associated up-regulation in hepatic leptin receptor expressionsuggested that hepatic leptin sensitivity was improved by thissupplement and this could, in part, contribute to reduced hepaticsteatosis.

Several studies have suggested that oxidative stress may play animportant role in the development of obesity-related complications suchas insulin resistance and type 2 diabetes. Consumption of red grapeextract improved fructose-induced ER-stress and insulin sensitivity inhealthy overweight first-degree relatives of type 2 diabetic patients.Similarly, our study revealed that supplementation with theflavonoid-rich ChrSd significantly improved insulin sensitivity andglucose metabolism, as shown by the 26% and 19% reductions in AUC during2-h ITT and GTT, respectively, as compared to the control. ChrSdsupplementation also significantly lowered the fasting glucoseconcentration. Additionally, this improved insulin sensitivity wasparalleled by down-regulation of hepatic Gdf15 expression. Gdf15expression has been reported to be induced in response to oxidativestress and inflammation, and its levels are increased in individualswith abdominal obesity, CVD, and insulin resistance. It has been shownthat suppression of the hepatic expression of genes related toinflammatory and immune processes was associated with improved liverfunction of HF-induced fatty liver. On the other hand, lipid derivativessuch as ceramides are a critical modulator of cellular stress and theiraccumulation inhibits insulin signaling. It has been shown thatflavonoids influence sphingolipid metabolism and are able to normalizethe elevated ceramide content of damaged liver cells. These observationsindicated a potential connection between the ChrSd-relateddown-regulation of hepatic Sptlc3, a gene related to ceramidebiosynthesis, and the reduced hepatic lipid content and improved insulinsensitivity reported in the present study. Therefore, improved insulinresistance and reduced hepatic lipid content appeared to be mediated bya reduction in oxidative stress and inflammation in DIO micesupplemented with ChrSd.

Glucokinase (Gck) activation may induce fatty liver in rodents becausethis enzyme phosphorylates glucose to produce glucose-6-phosphate; thisregulates hepatic glucose disposal and stimulates hepatic lipogenesis.Recent studies have shown that Gck overexpression in the liver increaseshepatic lipogenesis and circulating lipid concentrations. Furthermore,hepatic Gck expression was associated with hepatic lipogenic geneexpression and the lipid content of human liver biopsies. ChrSdsupplementation down-regulated hepatic expression of Gck and Gckr(glucokinase regulatory protein) and this may also have contributed toreducing the HF-induced hepatic lipid content.

In summary, it has been demonstrated herein that flavonoid-rich ChrSd, abyproduct of winemaking, improved HF-induced hepatic steatosis, plasmalipid profiles, and weight gain, as well as insulin resistance. Theseimprovements were associated with modulation of the hepatic expressionof genes related to bile acid, cholesterol, and fatty acid metabolism,oxidative stress, inflammation, and immune responses. Analysis of exonmicroarray data revealed that pathways involved in lipid and cholesterolmetabolism, and infectious and metabolic disease were differentiallyregulated by ChrSd. The decreased feed efficiency ratio, and reductionsin hepatic lipid content, adipose tissue weight, hepatic ceramidesynthesis, and oxidative stress may have contributed to the observedimprovement in insulin sensitivity. These results indicate thatconsumption of flavonoid-rich ChrSd can be beneficial for the preventionand/or treatment of NAFLD and other metabolic diseases due to itsreduction of oxidative stress and inflammation, modulation ofcholesterol and bile acid synthesis, modulation of lipid metabolism inliver, and amelioration of insulin resistance, as well as promotion ofoverall liver health.

TABLE 1 Diet Composition Ingredient (g/kg) Con ChrSd Lard fat 225.0225.0 Soybean oil 25.0 12.3 Cholesterol 0.8 0.8 MCC 52.6 18.6 Char seed0 100.0 Casein 200.0 182.5 Corn starch 145.6 109.8 Sucrose 300.0 300.0DL methionine 3.0 3.0 Choline bitartrate 3.0 3.0 Mineral mix 35.0 35.0Vitamin mix 10.0 10.0 Total weight 1000.0 1000.0 Calories/kg 4444 4427Con (Control diet containing 5% MCC, microcrystalline cellulose); ChrSdcontaining 10% Chardonnay grape seed flour

TABLE 2 Anthropometrics in DIO mice fed MCC and ChrSd for 5 wk¹ ConChrSd Body weight gain (g) 2.4 ± 0.6 −2.0 ± 0.7* Total energy intake(Kcal) 676.0 ± 13.3  784.1 ± 20.1* Feed efficiency ratio (g gain/g feed)0.18 ± 0.00 −0.13 ± 0.00* Liver weight (g) 1.6 ± 0.2  1.0 ± 0.1*Epididymal adipose tissue weight (g) 2.0 ± 0.1  1.3 ± 0.1* Hepaticpercent total lipid content 22.6 ± 2.2  12.8 ± 0.7* (g/100 g) ¹Valuesare means ± SE, n = 10. *indicate significant difference at P < 0.05.

TABLE 3 Summary of selected genes showing significant ≥|1.5|-foldhepatic modulation in mice fed a HF diet supplemented with ChrSdBiological Process Gene Fold- (GO)^(a)) Symbol Name Change Gene IDDown-regulated Activation of nuclear Chi311 Chitinase-like 1 −1.5NM_007695 factor-kappaB- inducing kinase activity Acyl-CoA metabolicCes1d Carboxylesterase 1D −1.6 NM_053200 process Adrenergic receptorAdra1b Adrenergic receptor, alpha −1.6 ENSMUST00000067258 signalingpathway 1b Aspartate transport Slc13a3 Solute carrier family 13 −1.7ENSMUST00000029208 (sodium-dependent dicarboxylate transporter), member3 Brain-derived Ntrk2 Neurotrophic tyrosine −3.4 ENSMUST00000079828neurotrophic factor kinase, receptor, type 2 receptor signaling pathwayC21-steroid hormone Cyp21a1 Cytochrome P450, family −1.6ENSMUST00000025223 biosynthetic process 21, subfamily a, polypeptide 1Canalicular bile acid Aqp8 Aquaporin 8 −1.8 NM_007474 transportCarbohydrate Gck Glucokinase −3.1 ENSMUST00000102920 metabolic processCarbohydrate Pfkm Phosphofructokinase, −1.6 NM_001163487 phosphorylationmuscle Cell redox Txn1 Thioredoxin 1 −1.5 NM_011660 homeostasis Cellularchloride ion Ckb Creatine kinase, brain −1.7 NM_021273 homeostasisCellular glucose Gckr Glucokinase regulatory −1.8 NM_144909 homeostasisprotein Cellular response to Fen1 Flap structure specific −2ENSMUST00000156291 DNA damage endonuclease 1 stimulus Cholesterolmetabolic Cyp46a1 Cytochrome P450, family −2.2 NM_010010 process 46,subfamily a, polypeptide 1 Vldlr Very low density −1.7ENSMUST00000167487 lipoprotein receptor Coenzyme A PpcdcPhosphopantothenoylcysteine −1.5 NM_176831 biosynthetic processdecarboxylase Degradation of ketone Bdh2 3-hydroxybutyrate −1.6NM_001172055 body dehydrogenase, type 2 Fatty acid biosynthetic Scd1Stearoyl-Coenzyme A −1.6 NM_009127 process desaturase 1 Fatty acidmetabolic Acot11 Acyl-Coenzyme A −2.2 NM_025590 process thioesterase 11Glucose homeostasis Mlxipl MLX interacting protein- −1.6 NM_021455(ChREBP) like Glucose and lipid Lcn13 Odorant binding protein −3.5ENSMUST00000077667 metabolism 2A Glutamate Prodh Proline dehydrogenase−1.6 ENSMUST00000003620 biosynthetic process Glutathione metabolic Gstt3Glutathione S-transferase, −1.7 NM_133994 process theta 3 G-proteincoupled Mc5r Melanocortin 5 receptor −1.6 NM_013596 receptor signalingTas2r104 Taste receptor, type 2, −1.5 NM_207011 pathway member 104Vmn1r192 Vomeronasal 1 receptor 192 −2.1 NM_145845 GTP catabolic processRasl2-9 RAS-like, family 2, locus 9 −2.2 NM_009028 Immune system process(Innate immune Cfd Complement factor D −2.4 NM_013459 response)(adipsin) (antigen processing Ctse Cathepsin E −1.7 NM_007799 andpresentation of exogenous peptide antigen via MHC class II) (Acute phaseOrm2 Orosomucoid 2 −1.7 NM_011016 response) (Regulation of Rorc Retinoicacid receptor- −1.5 NM_011281 gamma-delta T cell related orphan receptordifferentiation) gamma (Defense response Tlr5 Toll-like receptor 5 −2NM_016928 to bacterium) Methylation Hnmt Histamine N- −1.5 NM_080462methyltransferase Oxidation-reduction Cyp2b13 Cytochrome P450, family−3.4 NM_007813 process 2, subfamily b, polypeptide 1 Cyp2d40 CytochromeP450, family −1.8 ENSMUST00000055721 2, subfamily d, polypeptide 40 Hao2Hydroxy acid oxidase 2 −1.7 NM_019545 Stress-responsive Gdf15 Growthdifferentiation −2 ENSMUST00000003808 cytokine factor 15 SphingolipidSptlc3 Serine −1.7 ENSMUST00000110083 metabolic processpalmitoyltransferase, long (Ceramide de novo chain base subunit 3synthesis) Triacylglycerol Mogat1 Monoacylglycerol O- −1.6 NM_026713biosynthetic process acyltransferase 1 Triglyceride Plin4 Perilipin 4 −3NM_020568 metabolic process Up-regulated Adaptive immune Ifna9Interferon alpha 9 1.8 NM_010507 response (host immune defense)Asparagine Asns Asparagine synthetase 2.8 ENSMUST00000031766biosynthetic process Got1 Glutamate oxaloacetate 2 NM_010324transaminase 1, soluble ATP hydrolysis Atp6v0d2 ATPase, H+ transporting,2.2 ENSMUST00000029900 coupled proton lysosomal V0 subunit D2 transportBile acid Cyp7b1 Cytochrome P450, family 1.6 NM_007825 biosyntheticprocess 7, subfamily b, polypeptide 1 Cyp17a1 Cytochrome P450, family2.5 NM_007809 17, subfamily a, polypeptide 1 Bone morphogenetic Id1Inhibitor of DNA binding 1 1.7 NM_010495 protein signaling pathwayCalcium-mediated Avpr1a Arginine vasopressin 2.1 NM_016847 signalingreceptor 1A Cell-cell junction Ocln Occludin 1.5 ENSMUST00000069756organization Cholesterol Cyp51 Sterol 14-demethylase 5.6 NM_020010biosynthetic process Fdft1 Farnesyl diphosphate 1.9 NM_010191 farnesyltransferase 1 Hmgcr 3-Hydroxy-3- 2.5 NM_008255 methylglutaryl-Coenzyme Areductase Hsd17b7 Hydroxy steroid (17-beta) 2.8 NM_010476 dehydrogenase7 Insig1 Insulin induced gene 1 1.8 NM_153526 Lss Lanosterol synthase2.1 ENSMUST00000048678 Mvd Mevalonate (diphospho) 2.3 NR_028354decarboxylase Mvk Mevalonate kinase 2.2 ENSMUST00000043760 Nsdhl NAD(P)dependent 2 NM_010941 steroid dehydrogenase- like Sc4molSterol-C4-methyl oxidase- 4.5 ENSMUST00000034015 like Tm7sf2Transmembrane 7 1.7 NM_028454 superfamily member 2 Cholesterol effluxApom Apolipoprotein M 1.6 ENSMUST00000025249 Cholesterol Lepr Leptinreceptor 1.6 NM_146146 metabolic process Pcsk9 Proprotein convertase 1.9NM_153565 subtilisin/kexin type 9 Sqle Squalene epoxidase 13.5 NM_009270Cytokine-mediated Il17rb Interleukin 17 receptor B 1.5 NM_019583signaling pathway Fatty acid (β- Acsl3 Acyl-Coenzyme A 1.9 NM_028817oxidation synthetase long-chain family member 3 Glycine biosyntheticAgxt Alanine-glyoxylate 1.5 NM_016702 process, by aminotransferasetransamination of glyoxylate Glycolytic process Aldoc Aldolase C,fructose- 2.2 ENSMUST00000017534 bisphosphate Pgk2 Phosphoglyceratekinase 2 1.6 NM_031190 Inflammatory Tlr13 Toll-like receptor 13 1.6ENSMUST00000040065 response Lipid metabolic Hmgcs1 3-Hydroxy-3- 2.2NM_145942 process methylglutaryl-Coenzyme A synthase 1 Lipid transportStard4 StAR-related lipid transfer 1.9 ENSMUST00000025236 (START) domaincontaining 4 Proteinaceous Mmp7 Matrix metallopeptidase 7 1.5ENSMUST00000018767 extracellular matrix Regulation of Igfbp2Insulin-like growth factor 2.3 NM_008342 insulin-like growth bindingprotein 2 factor 1 receptor signaling pathway ^(a))Genes were classifiedinto biological process categories according to Gene Ontology Consortium(GO) classification.

TABLE 4 The top 10 biological functions and top 5 canonical and networkpathways of genes significantly modulated by ChrSd No. of genesBiological Function P value differentially expressed Lipid Metabolism 2.87 × 10⁻¹⁰- 48 3.11 × 10⁻² Small Molecule  2.87 × 10⁻¹⁰- 64Biochemistry 3.23 × 10⁻² Vitamin and Mineral  2.87 × 10⁻¹⁰- 21Metabolism 2.09 × 10⁻² Molecular Transport  2.52 × 10⁻⁷- 39 3.23 × 10⁻²Cell Morphology  6.92 × 10⁻⁴- 15 3.11 × 10⁻² Hematological Disease  5.48× 10⁻⁴- 13 3.12 × 10⁻² Metabolic Disease  5.48 × 10⁻⁴- 25 3.11 × 10⁻²Neurological Disease  6.17 × 10⁻⁴- 35 3.11 × 10⁻² PsychologicalDisorders  6.17 × 10⁻⁴- 27 3.11 × 10⁻² Dermatological Diseases  1.14 ×10⁻³- 18 and Conditions 1.05 × 10⁻² Canonical Pathway P value Ratio^(a))Superpathway of Cholesterol 1.81 × 10⁻¹⁷  12/87 (0.138) BiosynthesisCholesterol Biosynthesis I 1.53 × 10⁻¹³ 8/40 (0.2) CholesterolBiosynthesis II 1.53 × 10⁻¹³ 8/40 (0.2) (via 24,25-dihydrolanosterol)Cholesterol Biosynthesis III 1.53 × 10⁻¹³ 8/40 (0.2) (via Desmosterol)Zymosterol Biosynthesis 7.12 × 10⁻¹⁰  5/22 (0.227) Network PathwaySore^(b)) Lipid Metabolism, Small Molecule Biochemistry, Vitamin and 58Mineral Metabolism Infectious Disease, Organismal Injury andAbnormalities, Renal 36 and Urological Disease Protein Synthesis,Carbohydrate Metabolism, Lipid Metabolism 36 Lipid Metabolism, SmallMolecule Biochemistry, Vitamin and 33 Mineral Metabolism Behavior,Neurological Disease, Endocrine System 29 Development and Function Thefunctions and pathways that were most significant to the dataset wereidentified by Ingenuity Pathway Analysis (Ingenuity Systems).^(a))Number of molecules (genes) that met the cut-off criteria, dividedby the total number of molecules within a given pathway. ^(b))Likelihoodof finding the focus molecules in a given pathway, expressed as thenegative log of the P value.

While the embodiments have been depicted and described by reference toexemplary embodiments, such a reference does not imply a limitation onthe scope, and no such limitation is to be inferred. The embodiments arecapable of considerable modification, alteration, and equivalents inform and function, as will occur to those ordinarily skilled in thepertinent arts having the benefit of this disclosure. All referencescited herein are hereby incorporated by reference in their entirety andfor their intended purpose.

What is claimed is:
 1. A method of treating non-alcoholic fatty liverdisease (NAFLD) or non-alcoholic steatohepatitis (NASH) in a mammal inneed therefor, comprising administering to the mammal an amount of aChardonnay grape seed flour or a Chardonnay grape seed extract effectiveto treat NAFLD or NASH.
 2. The method of claim 1, wherein the Chardonnaygrape seed flour or a Chardonnay grape seed extract reduces the amountof fat deposited in the liver.
 3. The method of claim 1, wherein theChardonnay grape seed extract is a methanol or ethanol extract.
 4. Themethod of claim 1, wherein the Chardonnay grape seed flour or aChardonnay grape seed extract is administered in effective amount ofabout 1 mg to about 15,000 mg.
 5. The method of claim 1, wherein themammal is a human, companion animal, domestic pet, husbanded animal, orlivestock.
 6. The method of claim 1, wherein the method furthercomprises at least one additional compound.
 7. The method of claim 6,wherein the at least one additional compound is an antioxidant, ananti-cholesterol medication, anti-glycemic compound, or an insulinsensitizer.
 8. The method of claim 6, wherein the at least oneadditional compound is vitamin E, vitamin A, vitamin C, a carotenoid,lipoic acid, ubiquinol, ubiquinone, or glutathione.
 9. The method ofclaim 1, wherein the mammal is administered the Chardonnay grape seedflour.
 10. The method of claim 1, wherein the mammal is administered theChardonnay grape seed extract.
 11. A method of reducing hepaticsteatosis, steatohepatitis, or hepatic fibrosis in a mammal in needthereof, comprising administering to the mammal an amount of aChardonnay grape seed flour or a Chardonnay grape seed extract effectiveto reduce hepatic steatosis, steatohepatitis, or hepatic fibrosis. 12.The method of claim 11, wherein the mammal has been identified as havingnon-alcoholic fatty liver disease.
 13. A method of: reducing oxidativestress in a liver of a mammal in need thereof, comprising administeringto the mammal an amount of a Chardonnay grape seed flour or a Chardonnaygrape seed extract effective to reduce or prevent oxidative stress inthe mammal's liver; protecting, supporting, or enhancing, cytochromeP450 and/or Phase II enzymatic detoxification system in a liver of amammal in need thereof, comprising administering to the mammal an amountof a Chardonnay grape seed flour or a Chardonnay grape seed extracteffective to protect, support, or enhance cytochrome P450 and/or PhaseII enzymatic detoxification system in the mammal's liver; or ofincreasing production of antioxidants in a mammal, in need thereof, themethod comprising administering to the mammal an amount of a Chardonnaygrape seed flour or a Chardonnay grape seed extract effective toincrease the production of antioxidants in the mammal.
 14. The method ofclaim 13, wherein the method reduces or prevents lipid peroxidation. 15.The method of claim 13, wherein the antioxidant is glutathione,superoxide dismutase, or catalase.
 16. The method of claim 13, whereinthe mammal is administered the Chardonnay grape seed flour.
 17. Themethod of claim 13, wherein the mammal is administered the Chardonnaygrape seed extract.